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A  LABORATORY  MANUAL 

OF 


Physiological  Chemistry 


BY 


ELBERT  W.  ROCKWOOD,  M.D.,  Ph.D. 

PROFESSOR  OF  CHEMISTRY  AND  TOXICOLOGY  AND  HEAD  OF  THE  DEPARTMENT  OF  CHEMISTRY 

IN  THE  UNIVERSITY  OF  IOWA;  AUTHOR  OF  AN  INTRODUCTION  TO  CHEMICAL 

ANALYSIS  FOR  STUDENTS  OF  MEDICINE,  PHARMACY 

AND  DENTISTRY 


SECOND  EDITION,  REVISED  AND  ENLARGED 


TKHttb  ©ne  Colored  plate  anfc  Ubree  plates  of 
Microscopic  preparations 


PHILADELPHIA 
F.  A.  DAVIS  COMPANY,   PUBLISHERS 

1907 


COPYRIGHT,   1899 

BY 

THE  F.  A.  DAVIS  COMPANY 

COPYRIGHT,   1906 
BY 

F.  A.  DAVIS  COMPANY 


[Registered  at  Stationers'  Hall,  London,  Eng. 


-..... 

••"  •%•: 
•  •   •  •  • 


Philadelphia,  Pa.,  U.  S.  A. 

The  Medical  Bulletin  Printing  House 

1914-16  Cherry  Street 


PEEFACE  TO  THE  SECOND  EDITION. 


IN  no  department  of  biology  have  the  advances  of 
recent  years  been  more  marked  than  in  that  which  is 
studied  under  the  name  of  physiological  or  biological  chem- 
istry. While  the  subject  has  always  been  regarded  as  im- 
portant in  the  study  of  the  vital  processes,  its  value  is 
being  more  than  ever  emphasized  as  fundamental  in  a 
biological  or  medical  education.  A  revision  of  this  man- 
ual, with  the  addition  of  new  material,  has  therefore  seemed 
advisable.  No  change  has  been  made  in  the  original  plan, 
but  suggestions  arising  from  its  use  in  this  university  or 
from  the  experience  of  the  author's  colleagues  have  been 
adopted  wherever  they  promise  to  add  to  the  success  of 
the  work.  For  such  suggestions  particular  thanks  are  due, 
among  others,  to  Dr.  D.  W.  Fetterolf,  of  the  University  of 
Pennsylvania,  and  Dr.  Paul  Bartsch,  of  Howard  Univer- 
sity. 

ELBERT  W.  KOCKWOOD. 

THE  UNIVERSITY  OF  IOWA, 
1906. 


(iii) 


PEBFACE. 


IN  view  of  the  results  attained  from  the  course  given 
in  physiological  chemistry  in  this  University,  as  well  as 
the  experience  of  others,  the  author  is  firmly  convinced  of 
the  superiority  of  the  laboratory  method  of  instruction 
over  the  didactic,  believing  that  it  is  only  by  practical  work 
that  the  student  can  become  familiar  with  the  physio- 
logical changes  in  progress  in  the  animal  body  and  their 
products.  This  book  has  been  prepared  with  the  aim  of 
imparting  accurate  knowledge  through  the  student's  own 
observation.  It  has  seemed  advisable  to  include  with  the 
directions  for  experimental  work  a  brief  explanation  of 
the  facts  observed,  so  as  to  call  attention  to  their  meaning; 
or,  at  times,  to  state  others  which  are  important,  but  which 
could  not  well  be  demonstrated  in  such  a  course  as  this. 
Some  acquaintance  with  general  chemistry  and  with 
chemical  manipulation  is  presupposed." 

For  the  purpose  of  making  the  course  flexible,  the 
less  important  experiments,  or  those  which  are  not  of 
general  interest,  have  been  printed  in  smaller  type.  A 
few  blank  pages  have  been  inserted  for  additional  notes 
by  the  student.  It  has  been  found  that  the  time  usually 
assigned  to  chemistry  in  one  year  of  a  medical  course  is 
sufficient  for  the  performance  of  most  of  the  experi- 
mental work. 

As  far  as  possible,  the  work  has  been  so  arranged  as 
to  require  but  a  small  stock  of  apparatus  and  reagents 

(v) 


VI  PREFACE. 

and  such  as  are  readily  obtainable.  By  this  means  a  large 
class  can  carry  on  the  work  together.  Complicated  ex- 
periments have  been  omitted  or  put  in  small  type  for 
the  use  of  advanced  students  or  those  who  choose  to 
spend  more  time  upon  the  subject. 

The  animal  substances  which  are  required — albumin, 
blood,  bile,  and  others — can  be  found  in  the  market  or 
obtained  from  the  slaughter-house.  If  no  hospital  is 
near,  gastric  juice,  urine,  etc.,  corresponding  to  patho- 
logical specimens  can  be  prepared  artificially  for  testing 
by  the  student.  The  expense  of  the  course  is  very  small. 

ELBERT  W.  KOCKWOOD. 

UNIVERSITY  OF  IOWA, 
JULY  31,  1899. 


TABLE  OF  CONTENTS. 


PAGE 

INTRODUCTION  1 

THE  CARBOHYDRATES  1 

Starch   4 

Dextrin  8 

Glycogen    8 

Cellulose    11 

Glucose    12 

Lactose 10 

Sucrose   20 

Maltose    21 

Pentoses   21 

THE  FATS  23 

The  Lecithins 30 

THE  PROTEINS  32 

Albuminous  Substances    34 

Albumins    39 

Globulins 44 

The  Albuminates 46 

Proteoses  and  Peptones 48 

Fibrin  51 

Coagulated  Albumin    51 

Compound  Proteins   51 

THE  MUCINS   52 

The  Nucleoalbumins 53 

The  Nucleins   56 

Haematogen  58 

The  Albuminoids 58 

Collagen 58 

Gelatin    59 

Elastin  61 

Keratin    61 

FERMENTATION    62 

THE  SALIVA  66 

THE  GASTRIC  JUICE 71 

Gastric  Digestion  75 

Methods  of  Testing  Gastric  Juice 81 

(vii) 


Vlll  CONTENTS. 

PAGE 

THE  PANCREATIC  JUICE 93 

The  Pancreatic  Digestion 96 

Leucin  and  Tyrosin 99 

THE  BLOOD 101 

Composition   101 

The  Corpuscles    101 

The  Serum   106 

The  Coloring  Matters   '. 107 

Haemoglobin    107 

Oxyhsemoglobin Ill 

Methaemoglobin 113 

Hsematin  and  Hsemin 114 

Carbonic  Oxid  Haemoglobin 114 

Hsemochromogen    115 

Hsematoporphyrin 115 

Methods  of  Testing  Stains 121 

THE  BILE   122 

Biliary  Acids   125 

Cholesterin    128 

Biliary  Pigments   129 

CONNECTIVE  TISSUE 131 

White  Fibrous  Tissue  131 

Collagen 131 

Cartilage    132 

BONE  132 

MUSCULAR  TISSUE  134 

The  Plasma 138 

The  Extractives   139 

THE  BRAIN 141 

MILK 142 

THE  URINE  146 

Secretion  and  Physical  Properties 140 

Reaction  and   Fermentation 150 

Urea   152 

Uric  Acids  and  Urates 161 

Hippuric  Acid   165 

Creatinin    167 

Chlorids 167 

Phosphates   169 


CONTENTS.  IX 

PAGE 

Sulphates,  Inorganic  and  Organic 172 

Albuminuria 178 

Globulinuria  182 

Albumosuria 182 

Peptonuria    182 

Fibrinuria 184 

Glycosuria    184 

Acetonuria    186 

Diaceturia    187 

Laetosuria  188 

Clioluria   - 189 

Haemoglobinuria  and  Hsematuria   193 

Mucinuria  195 

Lipuria,  or  Chyluria   196 

URINABY  SEDIMENTS  197 

Classification    .  . ' 199 

Pus    200 

Mucus   200 

Epithelium    201 

Blood    202 

Casts   202 

Bacteria 205 

Spermatozoa    206 

Uric  Acid  and  Urates 207 

Calcium  Oxalate  208 

Phosphates   209 

Calcium  Sulphate 211 

Calcium   Carbonate    211 

Tyrosin    211 

Fat    212 

SYSTEMATIC  TESTING  OF  UKINE 212 

UBINABY  CALCULI  , .  215 

THE  METKIC  SYSTEM 218 

COMPOSITION  OF  REAGENTS  220 

INDEX  .  .  223 


PLATE  I. 


1.  tf,  Wheat-starch  granules. 
1),  Potato-starch  granules. 

2.  a,  Corn-starch  granules. 

b,  Buckwheat-starch  granules. 

3.  Hsemin  crystals,  color  brown. 

4.  Cholesterin,  colorless,  transparent. 

5.  Phenyl-glucosazone,  yellow. 

6.  a,  Urea,  colorless. 

6,  Urea  nitrate,  colorless. 


PLATE  I. 


PLATE  II. 


7.  Calcium  phosphate,  crystallized  and  amorphous  forms, 

both  colorless. 

8.  Triple  phosphate,  "coffin-lid"  crystals,  colorless. 
Sodium  urate,  brown  spherical  masses  with  spicules. 
Bacteria. 

9.  Ammonium  urate,  "thorn-apple"  forms,  color  brown. 
Calcium    carbonate,    spherules    and    dumb-bell    forms, 

colorless. 

10.  Calcium    oxalate,    "dumb-bell"    and    "envelope- shape" 

crystals,  colorless. 

11.  Uric  acid  crystals,  yellow  to  dark  brown. 
Amorphous  urates,  brownish. 

12.  o,  Calcium  sulphate  crystals,  colorless. 
6,  Impure  leucin,  nearly  colorless. 

c,  Tyrosin,  colorless  when  pure. 


PLATE  H. 


PLATE  III. 


13.  o,  Normal  pus-corpuscles   or  mucus-corpuscles,  gran- 

ular. 

b,  Pus-corpuscles  swollen   with   acetic   acid,   showing 

nuclei. 

c,  Pus-corpuscles    showing  amoeboid   movement.  .  All 

colorless. 

d,  Blood-corpuscles,  nearly  colorless. 

14.  Different  forms  of  epithelial  cells,  colorless. 

15.  Granular  casts,  colorless. 

16.  Epithelial  casts,  colorless. 

17.  Hyaline  casts. 

a,  Broad  or  waxy,  colorless. 

6,  Narrow,  colorless,  and  extremely  transparent. 

18.  a,  Fat-casts. 

6,  Yeast-fungi  in  urine. 
c,  Spermatozoa. 


PLATE  TIT. 


PLATE  IV. 


ABSORPTION-SPECTRA . 

1.  Oxyhsemoglobin. 

2.  Haemoglobin. 

3.  CO-hsBmoglobin  and  CO-hsemochromogen 

4.  Methsemoglobin,  alkaline. 

5.  Hoematoporphyrin,  acid. 

6.  Hsematoporphyrin,  alkaline. 

7.  Hsemochromogen,  alkaline. 

8.  Hsematin,  acid. 

9.  Hsematin,  alkaline. 

10.  Sulphur  metheemoglobin. 

11.  Methsemoglobin,  neutral  or  faintly  acid. 

12.  Pettenkofer's  test  for  biliary  acids. 


INTRODUCTION. 


THE  principal  materials  which  enter  into  the  com- 
position of  the  animal  body,  as  well  as  of  the  food  neces- 
sary for  its  support,  may  be  divided  into  several  general 

classes: — 

fl.  Water. 
I.  Inorganic  «  n    ,r.        ,      ,    . 

j  2.  Mineral  substances. 


^  1.  Non-nitrogenous       1  (a)  Carbohydrates. 
II    Organic  J       compounds,  such  as    j  (5)  Fats. 

"  I  2.  Nitrogenous    compounds,    such    as    the 
Proteins. 

There  are  a  number  of  nitrogenous  compounds  in  the 
animal  body  which  cannot  be  classed  under  the  proteins, 
and  others  which  contain  no  nitrogen,  but  which  do  not 
belong  to  the  carbohydrates  or  fats;  nevertheless,  these 
three  classes  include,  by  far,  the  largest  part  of  the  organic 
constituents. 


THE  CARBOHYDRATES. 

The  carbohydrates  are  composed  of  three  elements: 
carbon,  hydrogen,  and  oxygen.  The  latter  two  are  always 
present  in  proportion  to  form  water,  and  in  this  the  carbo- 
hydrates differ  from  the  fats,  which  contain  less  oxygen. 
The  name  of  the  group  is  derived  from  their  composition, 


THE   CAEBOHYDKATES. 


^  although  "tiiey  "cannot  be  made  directly  from  carbon  and 
water.  Most  of  them  contain  in  a  molecule  six  atoms  of 
carbon  or  some  multiple  of  six.  Many  organic  compounds 
of  carbon,  hydrogen,  and  oxygen,  however,  have  the  two 
latter  in  the  proportion  to  form  water,  but  do  not  belong  to 
the  carbohydrates. 

Carbohydrates  are  found  in  both  the  animal  and  vege- 
table kingdoms,  but  are  most  abundant  in  the  latter. 

The  different  members  of  the  group  differ  greatly  in 
their  properties,  such  as  the  power  of  crystallization,  fer- 
mentation, reducing  effect,  action  on  polarized  light,  taste, 
etc. 

They  may  be  divided  according  to  their  molecular 
composition  into  three  classes: — 

I.  Glucoses,  or  monosaccharids,  C6H1206,  including: 

1.  Glucose,  or  grape-sugar;  also  called  dextrose. 

2.  Fructose,  or  fruit-sugar;   also  called  laevulose. 

3.  Less  important  are  galactose,  mannose,  and  sev- 

eral others. 

II.  Saccharoses,   or   disaccharids,   C12H22011,   includ- 

ing:— 

1.  Sucrose,  or  cane-sugar. 

2.  Lactose,  or  milk-sugar. 

3.  Maltose,  or  malt-sugar,  and  some  others. 

III.  Amyloses,  or  polysaccharids,  (C6H1005)X,  includ- 
ing:— 

1.  Starch. 

2.  Dextrin. 

3.  Glycogen. 

4.  Cellulose;  also  a  number  of  gums  and  others  of 

less  importance. 


THE   CARBOHYDRATES.  3 

In  addition  to  the  above  classes  there  are  a  number  of 
compounds  containing  three,  four,  five,  seven,  eight,  or 
nine  atoms  of  carbon  in  a  molecule.  Their  chemical  names 
are  compounded  from  a  prefix  which  indicates  the  number 
of  carbon  atoms  with  the  suffix  ose,  thus,  triose,  tetrose, 
pentose,  etc.  As  yet  they  are  not  known  to  be  of  any  great 
importance  in  physiological  chemistry. 

The  first  class,  the  monosaccharids,  is  so  named  be- 
cause they  contain  one  of  the  groups  of  six  carbon  atoms. 
They  are  mostly  crystalline,  easily  soluble  in  water,  and 
have  a  sweet  taste.  Chemically  they  are  aldehyde  or  ketone 
compounds  of  the  hexatomic  alcohols.  The  former  are  in- 
dicated by  the  prefix  aldo-,  the  latter  by  keto-;  thus  aldo- 
pentose,  keto-hexose,  etc.  They  have  the  power  of  reduc- 
ing the  oxygen  compounds  of  the  metals  and  of  forming 
compounds  with  phenyl-hydrazin.  They  will  also  undergo! 
fermentation  with  yeast. 

The  disaccharids  contain  in  a  molecule  twelve  atoms  of 
carbon.  They  may  be  conceived  of  as  composed  of  two 
molecules  of  a  monosaccharid  minus  one  molecule  of  water: 

C6H1206  +  CGH1206  =  C12H220±1  +  H20. 

By  the  action  of  acids  or  ferments  they  take  up  a 
molecule  of  water  and  form  two  molecules  of  a  mono- 
saccharid. This  formation  of  one  or  more  simple  sugars 
from  a  molecule  of  a  disaccharid  is  called  inversion,  and 
the  resulting  sugar  is  known  as  invert-sugar.  The  di- 
saccharids do  not  undergo  fermentation  with  yeast  until 
they  have  been  inverted.  They  are  all  soluble  in  water  and 
have  a  sweet  taste. 

The  polysaccharids  contain  more  than  two  groups  of 
six  carbon  atoms,  though  the  number  is,  in  most  cases,  not 


4  THE   CARBOHYDRATES. 

positively  known,  and  therefore  is  represented  by  x.  They 
have  probably  a  much  higher  molecular  weight  than  the 
other  classes.  Their  constitution  is  not  known.  They  are 
mostly  amorphous,  insoluble  in  water,  and,  consequently, 
tasteless.  Ferments  and  acids  convert  them  into  the  mono- 
saccharids.  They  do  not  have  reducing  power. 

STARCH. 

Starch  occurs  in  the  cells  of  the  plant.  It  is  in  the 
form  of  grains  or  granules,  which  vary  in  size  in  different 
plants  from  about  0.002  of  a  millimeter  to  ten  times  that. 
The  granules  are  composed  of  two  parts:  an  inner,  soluble 
one,  called  granulose,  and  an  outer  one,  called  cellulose. 
This  latter  is  insoluble  in  water  and  protects  the  starch 
from  the  action  of  many  of  the  weaker  ferments.  When 
boiled  or  acted  upon  by  alkalies  it  is  broken,  allowing  the 
granulose  to  escape  and  forming  starch-paste,  or  soluble 
starch.  The  shape  and  size  of  the  granules  differ  so  much 
in  the  different  plants  that  the  source  can  often  be  deter- 
mined by  its  microscopic  appearance.  Those  of  the  potato 
have  a  shape  somewhat  similar  to  that  of  a  clam-shell,  those 
of  wheat  are  round  and  smaller,  and  those  of  buckwheat 
more  irregular.  (Plate  I,  1  and  2.) 

Starch  can  be  obtained  from  the  parts  of  the  plant 
where  it  is  stored  up,  like  the  tuber  of  the  potato  or  the 
kernel  of  grain,  by  macerating  it,  then  washing  out  the 
starch  with  cold  water. 

Starch  is  a  colloid.  A  colloid  is  a  substance  which 
when  dissolved  will  not  pass  through  an  animal  membrane 
or  parchment.  They  are  the  opposite  of  crystalloids,  which 
are  usually  crystalline  and  which  will  diffuse  through  such 
membranes.  This  process  of  diffusion  or  separation  of 
colloid  from  crystalloid  substances  is  called  dialysis.  As 


STAECH.  5 

starch  cannot  pass  through  an  animal  membrane,  it  must 
be  changed  to  a  diffusible  form  before  it  can  be  absorbed. 
This  is  effected  by  ferments  in  the  saliva  and  pancreatic 
fluid. 

By  heating  to  160°  to  200°  starch  is  converted  into 
dextrin.1  By  boiling  a  solution  with  a  dilute  acid  it  is 
changed  first  into  dextrin,  then  into  glucose.  Ptyalin 
changes  it  first  into  dextrin,  finally  to  maltose.  The  dias- 
tase of  malt  gives  the  same  products. 

Starch  gives  an  intense-blue  color  with  a  solution  of 
iodin.  This  color  disappears  on  heating  the  liquid;  but 
if  it  is  not  heated  too  long  it  becomes  blue  or  purple  again 
when  it  cools.  The  color  will  also  be  destroyed  by  the  ad- 
dition of  anything  which  will  form  a  compound  with  the 
iodin,  such  as  sodium  thiosulphate,  silver  salts,  or  the  alka- 
line hydrates. 

1. — Starch  may  be  prepared  from  a  potato  by  grating  it  upon 
a  tin  grater,  stirring  it  up  with  a  little  water,  and  squeezing  the 
water,  which  contains  a  large  part  of  the  starch,  through  a  piece 
of  unbleached  muslin.  After  repeating  this  with  several  portions 
collect  the  water  in  one  vessel  and  allow  the  starch  to  settle  to  the 
bottom.  Pour  off  the  water,  add  more,  and  allow  to  settle  again, 
repeating  till  the  starch  appears  clean  and  white.  Take  what  is 
needed  for  the  experiments  and"  let  the  rest  dry.  Enough  starch 
for  the  microscope  examination  can  be  obtained  from  the  scrap- 
ings from  a  potato  without  washing.  The  cellulose- fibers  will  then 
be  seen  also. 

2. — Examine  the  starch  under  the  microscope.     No-     -Y 
tice  the  shape  of  the  granules. 

3. — Place  a  drop  of  very  dilute  iodin  solution  upon 
the  slide  so  that  it  runs  under  the  cover-glass  and  notice 

1  Unless  otherwise  stated,  all  degrees  of  temperature  will  be 
understood  as  referring  to  the  centigrade  scale. 


6  THE   CARBOHYDRATES. 

the  markings  which  are  thus  brought  out  upon  the  gran- 
ules which  are  least  colored. 

4. — Examine  in  the  same  way  starch  from  other 
sources:  corn,  wheat,  buckwheat,  etc.  Observe  the  differ- 
ence in  the  size  and  shape  of  the  granules.  Sketch  these 
and  hand  in  the  results. 

5. — Prove  that  starch  does  not  dissolve  in  cold  water 
by  filtering  after  shaking  powdered  starch  in  a  test-tube  of 
water.  lodin  gives  no  color  to  the  filtrate. 

6. — Add  about  a  gramme  of  starch  to  100  cubic  centi- 
meters of  cold  water,  mix  it  thoroughly,  and  boil.  The 
starch  has  dissolved,  as  is  shown  by  filtering  and,  after 
cooling,  testing  a  portion  of  the  filtrate  with  iodin  solution. 
A  deep-blue  color  is  produced.  It  is  destroyed  by  heating, 
but  reappears  as  purple  or  blue  again  upon  cooling. 

V  7. — Use  a  piece  of  parchment  dialyzing  tube  to  test 
diffusibility.  First  see  that  this  does  not  leak.  It  should 
hold  water  when  suspended  by  the  two  ends.  Place  inside 
some  of  the  starch  solution  made  in  the  preceding  experi- 
ment and  hang  the  whole  in  a  small  beaker  of  water,  so 
that  the  liquids  inside  and  outside  are  at  the  same  level. 
ir  Instead  of  the  tube  a  piece  of  parchment  can  be  placed  in 
a  funnel  from  which  the  stem  has  been  broken,  as  if  the 
liquid  were  to  be  filtered.  Pour  the  starch  solution  into 
this  and  suspend  the  whole  in  a  beaker  of  water.  Allow 
it  to  stand  several  hours,  then  test  the  water  outside  with 
iodin  for  starch.  It  does  not  pass  through  because  it  is  a 
colloid.  Then  put  a  little  glucose  in  the  dialyzer.  It  dif- 
fuses out  and  can  be  found  by  Trommer's  reaction. 

8. — Examine  under  the  microscope  the  starch-paste 
which  has  been  made  by  heating  starch  in  water.  The 
granules  have  been  burst  open  and  destroyed. 


STAECH.  7 

9. — Prove  that  starch  can  be  decomposed  by  acids  or 
ferments  by  means  of  the  following: — 

In  about  100  cubic  centimeters  of  water  in  a  porce- 
lain dish  boil  enough  starch,  previously  moistened  with 
cold  water,  to  make  a  thin  paste.  Add  about  10  cubic  cen- 
timeters of  dilute  sulphuric  acid  and  boil,  stirring  at  first 
until  the  liquid  becomes  thinner.  Keep  the  solution  up  to 
its  original  volume  by  the  addition  of  water.  If  this  is  not 
done  the  strong  acid  will  turn  the  liquid  brown  or  black. 
From  time  to  time  remove  a  portion,  cool,  and  test  with 
iodin.  When  the  iodin  gives  a  red  color  the  starch  has 
been  converted  into  dextrin.  When  no  color  appears  on 
the  addition  of  iodin  it  has  been  changed  to  glucose.  Test 
a  portion  for  glucose  by  adding  an  equal  volume  of  sodium 
hydrate  solution,  then,  drop  by  drop,  cupric  sulphate  solu- 
tion till  a  deep-blue  color  is  produced.  Heating  this  will 
give  a  yellow  or  red  precipitate,  showing  the  presence  of  /? 
glucose.  This  is  known  as  Trommels  test  for  glucose. 

10. — To  a  clear  solution  of  starch  add  a  solution  of 
tannic  acid.  A  yellowish  precipitate  forms  which  dissolves 
on  heating. 

11. — Try  Trommer's  test  with  the  starch  solution.  It 
does  not  respond. 

*—  12. — Add  gradually  to  the  remainder  of  the  solution 
which  has  been  boiled  with  the  acid,  while  it  is  still  hot, 
powdered  calcium  or  barium  carbonate  until  it  is  neutral. 
Filter  and  evaporate  the  nitrate  to  dryness  on  the  steam- 
bath.1  Glucose  remains:  examine  its  properties  and  pre- 
serve it  for  subsequent  tests. 

1  To  evaporate  a  liquid  on  a  steam-  or  water-bath  the 
evaporating  dish  in  which  it  is  contained  can  be  heated  by  stand- 
ing it  on  a  beaker  of  boiling  water.  This  removes  all  danger  of 
burning  the  residue. 


THE   CARBOHYDRATES. 


DEXTRIN. 

Dextrin  is  the  intermediate  product  in  the  change  from 
starch  to  glucose  or  maltose.  There  have  been  several 
varieties  described:  erythrodextrin,  which  is  colored  red 
by  iodin;  achroodextrin,  which  is  not  so  colored,  etc. 

It  is  formed  from  starch  by  the  action  of  heat,  acids, 
or  ferments.  It  is  soluble  in  water,  making  a  sticky  liquid, 
often  used  for  a  mucilage.  It  is  produced  when  bread  is 
toasted,  and  is  also  found  in  the  crust.  Toast  or  bread- 
crust,  then,  has  its  starch  partially  changed  into  a  more 
diffusible  substance. 

13. — Prepare  dextrin  from  starch  by  heating  in  a  porcelain 
dish  on  a  sand-bath  half  a  spoonful  of  powdered  starch  previously 
dampened  with  a  few  drops  of  dilute  nitric  acid  (made  by  adding 
a  few  drops  of  nitric  acid  to  a  test-tubef ul  of  water) .  The  starch 
must  be  stirred  with  a  glass  rod  until  it  has  turned  yellowish  or 
brown,  when  it  has  been  changed  to  dextrin. 

14. — Dissolve  some  dextrin  in  water  and  test  with  a 
drop  of  iodin  solution.  A  red  or  brown  color  is  produced, 
not  a  blue,  if  the  change  has  been  complete.  If  commer- 
cial dextrin  is  tested  it  will  probably  be  found  to  contain 
undecomposed  starch. 

GrLYCOGEN.     yit 

Glycogen  is  found  in  a  few  of  the  lower  plants,  in  some 
shell-fish,  and  in  many  fluids  and  tissues  of  the  bodies  of 
mammals.  It  is  most  abundant  in  the  liver,  and  next  in 
the  muscles.  It  is  also  called  liver-sugar  or  liver-starch. 
In  the  animal  body  it  is  most  plentiful  when  the  animal  is 
well  nourished,  especially  after  a  full  meal.  At  such  times 
it  may  be  in  as  large  an  amount  as  10  or  12  per  cent,  of 


GLYCOGEN.  9 

the  liver,  but  it  is  usually  not  more  than  3  or  4  per  cent. 
It  disappears  completely  from  the  liver  after  long  starva- 
tion, or  more  quickly  through  severe  work  or  great  fright. 

It  is  best  obtained  from  the  liver.  After  boiling  to  kill 
the  ferments  which  are  always  present,  dissolving  in  water, 
and  removing  the  nitrogenous  substances,  it  can  be  pre- 
cipitated by  alcohol. 

Glycogen  is  an  amorphous,  white,  tasteless  powder. 
In  water  it  dissolves  to  an  opalescent  solution.  With  iodin 
it  gives  a  red  color,  which  disappears  on  heating.  It  does 
not  have  a  reducing  action  upon  cupric  hydrate.  Boiling 
with  acids  converts  it  into  dextrin,  then  maltose,  then  glu- 
cose. The  salivary  and  pancreatic  ferments  produce  the 
same  change. 

The  glycogen  of  the  liver  seems  to  be  formed  mostly 
from  the  carbohydrates  of  the  food,  but  partly,  at  least, 
from  the  nitrogenous  compounds. 

It  is  deposited  in  the  liver  as  a  reserve  material,  just 
as  the  starch  is  stored  for  a  reserve  material  in  the  plants. 
When  it  is  needed  by  the  body  it  is  converted  by  a  ferment 
into  grape-sugar,  and  this  passes  into  the  circulation.  It 
is  probable  that  it  is  used  to  furnish  energy  for  the  body. 
After  death  the  glycogen  quickly  disappears  from  the  tis- 
sues of  the  body,  being  decomposed  by  the  ferments  which 
are  present.  If  these  are  destroyed  by  boiling  the  tissue 
for  a  short  time  the  glycogen  is  not  destroyed,  but  can  be 
extracted. 

15.  PREPARATION  OF  GLYCOGEN. — In  a  mortar  grind  with 
sand  or  glass  about  25  grammes  of  the  adductor  muscle  of  the 
scallop  (pecten  irradiens),  extract  several  times  with  50  cubic  centi- 
meters of  cold  water,  repeating  the  operation  with  hot  water. 
Boil  the  liquid  to  coagulate  the  proteins,  filter  and  concentrate  the 
nitrate  to  about  50  cubic  centimeters,  then  add  alcohol  to  70  per 
cent,  in  strength.  This  precipitates  the  glycogen.  Filter  this  off. 


10  THE   CARBOHYDRATES. 

If  the  dry  powder  is  desired,  wash  with  alcohol,  then  with  ether, 
and  dry  in  a  desiccator. 

Prepare  glycogen  from  the  liver  of  a  freshly-killed,  well-nour- 
ished animal.  The  animal  is  best  killed  while  digestion  is  in 
progress.  If  a  rabbit,  this  may  be  an  hour  after  introducing  10  to 
15  grammes  of  sugar  into  the  stomach  through  a  tube.  Remove 
the  liver  as  soon  as  posssible,  cut  it  into  lumps,  and  immediately 
put  it  into  about  four  times  its  weight  of  boiling  water.  Let  it 
boil  half  an  hour,  then  rub  up  the  lumps  as  much  as  possible  in  a 
large  mortar,  add  water,  and  boil  again.  Filter  through  muslin, 
concentrate  upon  the  water-bath  to  about  one-fourth  its  volume, 
and  allow  the  solution  to  cool.  Then  precipitate  the  gelatin  and 
other  protein  compounds  by  adding  alternately  small  quantities  of 
hydrochloric  acid  and  potassio-mercuric  iodid1  as  long  as  anything 
is  thrown  down.  Filter  and  add  to  the  filtrate  twice  its  volume  of 
alcohol  to  precipitate  the  glycogen.  Wash  with  alcohol.  To  purify 
the  substance  it  should  be  dissolved  in  a  little  water  and  precipi- 
tated again  with  alcohol.  If  the  anhydrous  powder  is  desired,  the 
water  must  be  removed  as  far  as  possible  before  drying.  To  accom- 
plish this  wash  the  precipitate  next  with  absolute  alcohol,  then 
with  ether  to  remove  the  alcohol.  Dry  in  a  vacuum-desiccator  over 
sulphuric  acid.  If  the  pure  substance  is  not  desired,  the  tests  may 
be  made  on  the  solution  after  the  removal  of  the  protein  com- 
pounds. 

1.6. — If  the  dry  substance  has  been  obtained,  try  its 
taste  and  its  solubility  in  water.  Test  the  solution  with 
iodin.  It  gives  a  red  color. 

17.— Try  Trommer's  test  (Experiment  9).  There  is 
no  red  color  if  the  glycogen  has  been  purified.  If  it  has 
not  been  it  contains  glucose,  which  responds  to  the  test. 

18. — Convert  one  portion  of  the  solution  into  glucose 
by  heating  with  hydrochloic  acid  and  another  by  the  action 
of  saliva.  Test  each  for  the  glucose  by  Trommer's  test. 

19. — Prove  that  the  glycogen  is  destroyed   (changed 

Prepare  by  precipitating  mercuric  chlorid  with  potassium 
iodid,  washing  the  precipitate  and  then  adding  it  to  a  hot  solu- 
tion of  potassium  iodid  as  long  as  it  dissolves. 


CELLULOSE.  11 

to  a  reducing  sugar)  in  the  liver  after  death  by  the  action 
of  a  ferment,  making  the  test  upon  some  liver  from  the 
market.  (Instead  of  this  a  part  of  the  liver  from  Experi- 
ment 14  can  be  used.  This  should  be  after  it  has  stood 
several  hours  in  a  warm  place.)  Chop  it  finely  and  extract 
with  boiling  water.  Acidify  the  solution  slightly  with 
acetic  acid,  add  a  little  sodium  chlorid,  and  boil  to  pre- 
cipitate the  protein  compounds.  After  filtering,  test  the 
filtrate  for  glycogen  by  means  of  iodin  and  also  for  sugar 
by  Trommer's  test. 

20. — Add  a  little  blood  to  a  test-tube  of  the  glycogen 
solution  and  after  it  has  stood  ten  minutes  in  a  beaker  of 
water  at  body-temperature  slightly  acidify  with  acetic  acid, 
boil,  and  filter  to  remove  the  albumin,  and  test  the  filtrate 
for  glucose  and  glycogen.  The  latter  has  been  converted 
into  glucose  by  a  ferment  which  is  found  in  the  blood. 

CELLULOSE. 

Cellulose  forms  the  membrane  of  the  plant-cells,  and 
is  not  found  as  a  constituent  of  the  animal  body,  except  in 
a  few  of  the  lower  forms.  Cotton  and  filter-paper  are  two 
of  the  most  common  examples.  It  is  distinguished  from 
the  other  polysaccharids  by  its  insolubility.  It  is  insoluble 
in  the  ordinary  solvents,  but  can  be  dissolved  in  the  strong 
mineral  acids,  being  converted  into  dextrin.  It  also  dis- 
solves in  a  solution  of  cupric  hydrate  in  ammonia. 
(Schweitzer's  reagent),  and  in  a  solution  of  zinc  chlorid 
(Schultze's  reagent).  Sulphuric  acid  changes  paper  into  a 
parchment-like  substance  by  covering  the  surface  with  a 
coating  of  its  decomposition-products  and  so  sticking  the 
fibers  together.  Iodin  does  not  stain  the  unaltered  cellu- 
lose, but  does  so  after  it  has  been  acted  upon  by  the  acid. 


12  THE   CARBOHYDRATES. 

Cellulose  is  only  slightly  attacked  by  the  digestive  ferments 
of  man,  though  the  herbivorous  animals  digest  it  to  a 
greater  extent.  By  the  continued  action  of  acids  it  is  con- 
verted into  glucose. 

21. — Show  that  cellulose  is  not  stained  by  iodin.  Use 
absorbent  cotton  or  starch-free  filter  paper. 

_22.-=Try  the  solubility  of  cotton  or  filter-paper  in  solution  of 
zinc  chlorid  (Schultze's  reagent)  and  also  in  a  solution  of  cupric 
hydrate  in  ammonium  hydrate  (Schweitzer^s  reagent).  It  can  be 
precipitated  from  these  solutions  by  dilution  with  water. 

23. — To  one  volume  of  water  in  a  beaker  add  slowly 
two  volumes  of  concentrated  sulphuric  acid,  stirring  mean- 
while. Cool  the  mixture;  then  immerse  in  it  for  a  few 
seconds  a  piece  of  heavy  filter-paper,  plunging  it  into  a 
large  beaker  of  cold  water  as  soon  as  it  is  removed.  If  the 
time  of  immersion  has  been  correct  it  will  be  semi-trans- 
parent after  washing,  and  as  tough  as  an  animal  mem- 
brane. It  is  called  vegetable  parchment.  It  can  be  stained 
blue  by  iodin. 

24. — Let  another  piece  of  paper  remain  in  a  small 
amount  of  the  warm  acid  until  it  has  entirely  disappeared. 
Then  dilute  a  little  of  the  acid  with  water  and  test  it  for 
glucose  by  Trommer's  test  (Experiment  9),  being  sure  that 
enough  alkali  has  been  added  to  give  it  an  alkaline  reac- 
tion. 

GLUCOSE  (C6H1206). 

Glucose  is  also  called  dextrose  and  grape-sugar.  It  is 
found  in  the  vegetable  kingdom  as  well  as  in  the  animal. 
It  is  normally  present  in  the  blood  and  lymph  and  in  other 
fluids  of  the  body.  Pathologically  it  is  found  in  consider- 
able quantities  in  the  urine,  sometimes  in  as  large  amounts 
as  10  per  cent,  or  more.  The  urine  may  also  temporarily 


GLUCOSE.  13 

contain  grape-sugar  after  a  diet  rich  in  carbohydrates. 
Whether  it  may  normally  occur  in  very  small  amounts  in 
the  urine  is  a  question  which  is  often  discussed,  but  upon 
which  there  is  no  general  agreement. 

Glucose  is  made  commercially  by  boiling  starch  with 
a  dilute  acid.  It  can  be  produced  from  any  of  the  poly- 
saccharids  or  disaccharids  in  the  same  manner.  They  unite 
with  one  or  more  molecules  of  water,  forming  glucose: — 

(C6H1005)x+x  H20  =  x  C6H1206. 
C12H2  Ai  +  H20  —  2C6H1206. 

Pure  glucose  can  be  made  from  pure  cane-sugar  by 
dissolving  it  in  alcohol  and  adding  hydrochloric  acid.  The 
glucose  crystallizes  out  on  standing. 

Glucose  is  a  crystalline  substance,  but  crystallizes  with 
difficulty  from  water.  It  can  better  be  crystallized  from 
methyl  alcohol  or  ethyl  alcohol.  Its  taste  is  sweet,  but  less 
so  than  that  of  cane-sugar.  It  is  easily  soluble  in  water  or 
hot  alcohol.  With  yeast,  glucose  ferments  best  at  about 
25°  C.,  forming  alcohol  and  carbon  dioxid: — 

C6H1206  =  2C2H5OH  +  2C02. 

In  the  presence  of  milk  or  cheese  it  ferments  to  lactic 
acid.  Calcium  carbonate  or  oxid  of  zinc  must  be  added  to 
keep  the  solution  neutral  if  it  is  desired  that  the  action  go 
on  for  a  long  time,  as  the  presence  of  the  acid  kills  the 
ferment: — 

C6H1206  =  2C3H603. 

By  the  action  of  another  ferment  the  lactic  acid  is 
changed  into  butjrric  acid: — 

2C3H603  =  C4H802  +  2C02  +  4H. 


14  THE   CARBOHYDRATES. 

25. — Prepare  pure  glucose  from  cane-sugar  by  the  following 
method: — 

Acidify  100  cubic  centimeters  of  90-per-cent.  alcohol  with 
4  cubic  centimeters  of  concentrated  hydrochloric  acid,  warm  the 
liquid  upon  the  water-bath  to  45°,  and  add  gradually  30  grammes 
of  finely-powdered  cane-sugar,  stirring  until  it  has  dissolved.  The 
temperature  should  not  rise  above  50°.  After  two  hours  at  50° 
the  sucrose  has  been  inverted.  Then  let  it  stand  in  a  cool  place. 
The  glucose  commences  to  crystallize  out  in  about  a  week,  but 
crystallization  may  be  hastened  by  adding  to  the  cold  solution  a 
few  crystals  of  glucose  and  by  frequent  stirring.  After  the  glucose 
has  crystallized  from  the  solution  filter,  best  with  the  aid  of  a 
filter-pump;  wash  free  from  the  acid  by  90-per-cent.  alcohol,  then 
by  absolute  alcohol;  finally  dry  the  crystals.  It  may  be  purified 
by  dissolving  in  pure  methyl  alcohol  by  the  aid  of  heat  and 
allowing  it  to  again  crystallize  out. 

26. — Prove  that  cupric  hydrate  (made  by  the  addi- 
tion of  a  few  drops  of  copper  sulphate  to  a  sodium  hydrate 
solution)  is  soluble  in  a  solution  of  glucose,  giving  a  deep- 
blue  liquid.  Also  show  that  this  blue  solution  of  copper 
and  sugar  is  decomposed  by  heating,  and  yellow  or  red 
precipitate  of  cuprous  oxid  is  produced.  This  is  Trom- 
mels test  for  grape-sugar.  To  perform  the  test,  mix  about 
equal  volumes  of  sodium  hydrate  or  potassium  hydrate  and 
the  glucose  solution,  then  drop  in  copper  sulphate  solution 
until  a  permanent  precipitate  begins  to  form  or  until  the 
mixture  is  deep  blue ;  finally,  heat  the  solution. 

27. — Show  that  cupric  hydrate  is  also  soluble  in  a 
solution  of  Rochelle  salt  or  glycerin  in  water  if  an  alkaline 
hydrate  is  present,  but  that  these  solutions  are  not  decom- 
posed by  boiling.  Add  to  each  of  them  a  little  grape-sugar 
and  heat.  Cuprous  oxid  is  formed  in  both  cases.  The 
former  is  called  Fehling's  test,  the  latter  Haines's  test  for 
glucose. 


GLUCOSE   TESTS.  15 

28. — Show  that  a  glucose  solution  will  reduce  copper 
acetate  acidified  with  acetic  acid1  when  heated  for  some 
time  in  a  water-bath.  This  is  Barfoed's  test.  Observe  the 
difference  between  glucose  and  lactose  with  this  test. 

29. — Prove  that  when  heated  alone  in  water  cupric 
hydrate  gives  the  black  cupric  oxid  and  not  the  red  cuprous 
oxid;  also  that  with  an  excess  of  the  copper  solution  the 
black  may  hide  the  red  oxid  if  only  a  small  amount  of 
sugar  is  present. 

30. — Show  that  glucose  will  also  reduce  the  sub- 
nitrate,  or  basic  nitrate,  of  bismuth  if  its  solution  is  made 
alkaline  by  sodium  hydrate  or  carbonate  and  boiled  with 
the  bismuth  compound.  The  bismuth  oxid  which  is  formed' 
is  a  black  powder,  but  if  mixed  with  much  of  the  unre- 
duced bismuth  subnitrate  it  may  appear  gray.  This  is 
Boettger's  test.  The  bismuth  subnitrate,  like  cupric  hy- 
drate, is  soluble  in  an  alkaline  solution  of  Eochelle  salt, 
and  this  solution  when  heated  with  glucose  gives  the  black 
oxid  as  a  precipitate  (Nylander's  test). 

Heat  a  little  of  the  bismuth  subnitrate  in  a  solution  of 
albumin  which  has  been  made  strongly  alkaline  with  sodium 
hydrate,  and  notice  that  the  sulphid  of  bismuth,  which  is  formed, 
has  the  same  appearance  as  the  black  oxid  which  is  produced  by 
the  glucose;  that  is,  albumin  gives  a  similar  result  to  that  obtained 
with  grape-sugar. 

31. — Dissolve  in  a  small  amount  of  water  as  much 
phenyl-hydrazin  hydrochlorid  as  can  be  taken  up  on  the 
point  of  a  knife-blade  and  twice  as  much  sodium  acetate, 
filtering  if  it  is  not  clear.  Add  it  to  half  a  test-tubeful  of 
the  sugar  solution,  place  the  tube  in  a  beaker  of  boiling 


xThe  mixture  of  solution  and  sugar  solution  should  contain 
1.0  per  cent,  of  cupric  acetate  and  1.0  to  1.25  per  cent,  of  acetic  acid. 


16  THE   CABBOHYDRATES. 

water,  and  heat  it  for  an  hour.  Then  cool  it  and  examine 
the  precipitate  with  the  microscope.  It  is  phenyl-gluco- 
sazon :  bright-yellow,  needle-shaped  crystals.  They  may  be 
single,  but  are  more  often  in  clusters  (Plate  I,  5).  They 
can  be  distinguished,  if  necessary,  from  similar  compounds 
of  the  other  sugars  by  their  melting-point,  which  is  204°  C. 
If  they  separate  in  the  amorphous  state,  they  may  be  crys- 
tallized, after  filtering,  by  dissolving  in  a  little  hot  alcohol, 
then  evaporating  the  alcohol  to  a  small  volume,  and  letting 
it  stand.  Make  sketches  of  these :  hand  them  in. 

32. — Crush  a  piece  of  condensed  yeast  as  large  as  a 
pea  in  a  test-tube  of  water,  and  wash  it  two  or  three  times 
by  decantation  to  remove  any  fermentable  substances  which 
may  be  present.  Fill  the  tube  completely  full  of  a  glu- 
cose solution.  Mix  and  place  it,  still  full  of  the  liquid, 
with  the  mouth  downward  in  a  beaker  which  contains  a 
little  water,  or,  better,  some  of  the  grape-sugar  solution. 
Let  it  stand  for  twenty-four  hours  in  a  warm  place.  The 
carbon  dioxid,  which  is  formed.,  is  found  in  the  test-tube 
and  the  alcohol  in  the  liquid.  The  gas  may  be  proved  to 
be  carbon  dioxid  by  shaking  it  with  lime-water,  which  it 
turns  white.  The  presence  of  the  alcohol  is  shown  by 
warming  the  liquid  after  the  addition  of  sodium  hydrate 
and  a  little  iodin.  lodoform  separates  out  in  yellow  scales, 
or,  if  the  amount  of  alcohol  is  very  small,  the  odor  alone 
may  be  perceived. 

A  convenient  piece  of  apparatus  for  carrying  on  this 
fermentation  is  the  saccharimeter.  This  is  essentially  a 
graduated  tube  with  bulb  to  hold  the  liquid  which  is  forced 
out  by  the  gas.  From  its  reading  the  amount  of  glucose 
can  be  learned. 


GLUCOSE.  17 


QUANTITATIVE   TEST   FOR   GLUCOSE. 

Fehling's  Method. — The  solutions  used  are:  (A)  34.64 
grammes  of  cupric  sulphate  (CuS04,  5H20),  dissolved  in 
enough  water  to  make  the  volume  500  cubic  centimeters. 
The  crystals  used  must  be  dark  blue  and  not  effloresced; 
(B)  187  grammes  of  pure  Eochelle  salt  and  68  grammes  of 
sodium  hydrate  in  water  enough  to  make  the  volume  500 
cubic  centimeters.  These  solutions  must  be  kept  separate. 

33. — For  each  determination  mix  5  cubic  centimeters 
of  A  with  5  cubic  centimeters  of  B,  measuring  carefully 
with  a  pipette.  Add  about  40  cubic  centimeters  of  water, 
and  heat  to  boiling  in  a  beaker  or  porcelain  dish.  If  the 
solution  is  good  there  will  be  no  red  precipitate. 

The  best  results  are  obtained  when  the  solution  con- 
tains from  0.5  per  cent,  to  1.0  per  cent,  of  sugar;  that  is, 
when  from  5  to  10  cubic  centimeters  are  necessary  to 
destroy  the  blue  color  of  the  Fehling  solution.  If  it  con- 
tains more  than  this,  it  must  be  diluted  with  water  to  5  or 
10  times  its  volume,  measuring  accurately  the  water  added 
and  mixing  thoroughly. 

The  Fehling  solution  after  dilution  is  heated  to  boil- 
ing, and  the  sugar  solution  run  in  from  a  burette  until  the 
blue  color  has  been  destroyed,  leaving  the  liquid  colorless 
above  the  red  precipitate.  If  too  much  sugar  has  been 
added  it  begins  to  turn  yellowish.  The  amount  of  sugar 
is  ascertained  most  quickly  by  making  two  determinations: 
first,  a  rough  one,  then  one  which  is  made  more  carefully. 
Make  the  first  by  running  in  the  sugar  solution,  2  or  3 
cubic  centimeters  at  a  time,  as  long  as  the  blue  color  is 
well  marked,  then  1  cubic  centimeter  at  a  time,  heating  to 
boiling  after  each  addition.  It  can  be  learned  by  this  first 
test  within  1  or  2  cubic  centimeters  how  much  will  be  re- 


18  THE  CARBOHYDRATES. 

quired.  Then  rinse  out  the  beaker,  take  again  10  cubic 
centimeters  of  the  Fehling  solution,  diluted  as  before;  heat 
to  boiling  and  run  in  at  once  within  1  cubic  centimeter  of 
the  necessary  amount  of  the  sugar  solution.  Bring  it  to  a 
boil.  Then  add  the  sugar  solution  a  few  drops  at  a  time, 
heating  after  each  addition,  until  the  blue  color  has  just 
been  decolorized. 

Since  10  cubic  centimeters  of  the  Fehling  solution  is 
decolorized  by  0.05  gramme  of  glucose,  the  amount  of  the 
sugar  solution  or  urine  which  has  been  used  from  the 
burette  must  have  contained  0.05  gramme  of  glucose.  Eead 
the  volume  which  has  been  poured  from  the  burette,  and 
calculate  the  percentage  of  sugar  in  the  original  solution. 
If  this  has  been  diluted  with  water  the  amount  in  the  dilute 
solution  must  be  multiplied  by  the  number  of  times  it  was 
diluted.  Eemember  that  however  much  of  the  sugar  solu- 
tion may  have  been  used  to  destroy  the  blue  color,  it  con- 
tained 0.05  gramme  of  sugar.  For  example,  if  the  amount 
used  was  10  cubic  centimeters,  there  would  be  0.005 
gramme  of  glucose  in  1  cubic  centimeter;  that  is,  in  1 
gramme  of  solution.  In  100  grammes  there  would  be  0.5 
gramme  of  glucose,  or  0.5  per  cent. 

The  floating  red  precipitate  of  cuprous  oxid  obscures  the  encl 
reaction  and  makes  the  titration  slow  if  time  is  allowed  for  this 
to  settle.  Purdy's  modification  of  Fehling's  reagent  consists  in 
the  addition  of  a  large  excess  of  ammonium  hydrate  which  pre- 
vents the  precipitation  of  the  cuprous  oxid.  The  reagent  con- 
tains:— 

Cupric  sulphate 4.742  grm. 

Glycerin,  pure 38  cc. 

Dissolve  in  about  200  cc.  of  water. 

Potassium  hydrate 23.5  grm. 

Dissolve  in  about  200  cc.  of  water. 


LACTOSE.  19 

When  these  solutions  have  cooled  mix  them,  add  450  cubic 
centimeters  of  concentrated  ammonia  (sp.  gr.  0.9)  and  dilute  to 
1000  cubic  centimeters. 

For  the  titration  use  35  cubic  centimeters  copper  solution 
diluted  with  about  twice  its  volume  of  water.  Heat  this  to  boil- 
ing, then  drop  in  slowly  from  a  burette  the  urine  or  glucose  solu- 
tion until  the  blue  has  been  destroyed.  Three  to  five  seconds 
should  elapse  between  the  drops.  When  this  has  occurred  0.02 
gramme  of  glucose  has  been  added.  The  solution  should  then  be 
colorless.  If  it  is  yellow  an  excess  of  sugar  has  been  added.  From 
the  volume  of  solution  used  calculate  the  per  cent,  of  glucose  by 
weight. 

LACTOSE  (MILK-SUGAR:  C12H2201;l  +  H20). 

Lactose  is  found  in  the  milk  of  all  mammals  and  occa- 
sionally during  pregnancy  in  the  urine.  It  can  be  obtained 
from  the  milk  by  crystallization  after  the  removal  of  the 
nitrogenous  constituents. 

It  is  a  crystalline  substance,  soluble  in  water,  with  a 
faint  sweetish  taste.  With  pure  yeast  it  does  not  ferment. 
By  the  action  of  certain  other  ferments,  however,  it  under- 
goes alcoholic  fermentation,  with  the  production  at  the 
same  time  of  lactic  acid,  forming  the  drinks  known  as 
<rkoumiss,"  when  made  from  mares'  milk,  and  "kephyr" 
when  from  cows'  milk.  The  ordinary  souring  of  milk  is 
due  to  the  formation  of  lactic  acid  from  the  lactose  by 
micro-organisms. 

Milk-sugar  gives  with  many  reagents  the  same  results 
as  glucose.  It  can  be  distinguished  from  glucose  by  its  not 
fermenting  with  yeast  and  by  its  having  a  less  strong  power 
of  reduction,  it  is  unable  to  reduce  cupric  compounds  to 
cuprous  in  acetic  acid  solutions  (Barfoed's  test). 

34.  PREPARATION  OF  MILK-SUGAR.  —  Dilute  200 
cubic  centimeters  of  milk  with  800  cubic  centimeters  of 
water,  and  add  very  cautiously  not  more  than  0.1  per  cent. 


20  THE   CARBOHYDRATES. 

of  acetic  acid  to  precipitate  the  casein  (when  enough  has 
been  added  the  liquid  is  nearly  clear).  Filter.  Boil  the 
nitrate  and  filter  off  the  .coagulated  albumin.  Evaporate 
the  filtrate  upon  a  water-bath  to  a  syrup  and  allow  it  to 
stand  until  the  sugar  has  crystallized  out.  It  may  be  puri- 
fied by  recrystallizing  it. 

35. — Test  the  milk-sugar  with  Trommels,  Fehling's, 
and  the  phenyl-hydrazin  tests,  and  notice  that  the  results 
are  similar  to  those  obtained  with  glucose. 

36. — Try  Barfoed's  and  the  fermentation  tests  as 
made  with  the  glucose,  and  observe  that  the  results  are 
negative. 

37. — Boil  the  milk-sugar  solution  with  a  little  hydro- 
chloric acid,  neutralize,  and  try  the  fermentation  or  Bar- 
foed's  test.  Glucose  has  been  formed  and  this  will  now 
ferment,  forming  carbon  dioxid  and  alcohol. 

SUCROSE  (CANE-SUGAR:  C12H22011). 

Cane-sugar  is  found  in  plants,  not  in  the  animal  king- 
dom. It  has  no  reducing  power  and  does  not  respond  to 
the  tests  where  such  a  reducing  action  occurs,*  such  as 
Trommer's,  Fehling%  and  Boettger's.  It  is  decomposed 
by  heating  with  acid  into  a  molecule  of  glucose  and  one 
of  fructose. 

38. — Apply  Trommer's  or  Fehling's  test  to  a  solution 
of  pure  cane-sugar.  It  gives  no  results. 

39. — Boil  a  solution  of  cane-sugar  with  a  little  sul- 
phuric or  hydrochloric  acid,  neutralize  the  solution,  and 
prove  that  it  contains  glucose. 

40. — The  "invert  sugar"  which  results  from  the  decomposition 
of  cane-sugar  by  acids  can  be  separated  into  glucose  and  fructose 
by  adding  to  10  parts  6  parts  of  calcium  hydrate  and  50  parts  of 
water.  Both  sugars  form  calcium  compounds.  That  with  glucose, 


MALTOSE.  PENTOSES.  21 

being  liquid,  can  be  pressed  out  of  the  fruit-sugar  compound, 
which  can  then  be  decomposed,  the  fruit-sugar  being  set  free,  by 
the  addition  of  oxalic  acid  as  long  as  a  precipitate  is  produced. 
Filter  and  obtain  the  fructose  by  the  evaporation  of  the  filtrate. 

MALTOSE  (MALT-SUGAE:  C^H^O^,  H20). 

41. — Boil  a  small  lump  of  starch  with  25  cubic  centi- 
meters of  water,  cool  it  to  nearly  body  temperature,  and 
add  an  aqueous  extract  of  ground  malt,  made  at  the  same 
temperature.  Observe  that  the  mixture  becomes  thinner 
and  that  finally  a  sample  is  not  turned  blue  by  iodin.  Then 
test  for  the  maltose  with  phenyl-hydrazin  as  in  the  glucose 
reactions.  Try  also  Trommels  test  or  Fehling's  test.  It 
responds  to  all. 

42.  PREPARATION  OF  PURE  MALTOSE. — One  hundred  grammes 
of  starch  are  to  be  mixed  with  500  cubic  centimeters  of  cold  water 
as  thoroughly  as  possible,  then  heated  on  a  water-bath  until  it 
makes  a  paste.  Make  a  watery  extract  of  malt  at  40°  C.  from  6 
or  7  grammes  of  dry  malt.  When  the  starch-paste  has  cooled 
down  to  60°  or  70°,  add  the  malt-extract  and  keep  it  at  this  tem- 
perature for  an  hour.  When  the  starch  has  been  converted  to 
maltose  the  liquid  becomes  thin  and  watery.  Then  boil,  filter, 
and  evaporate  to  a  syrup  upon  the  water-bath.  Dissolve  the 
maltose  from  the  residue  with  small  portions  of  90-per-cent. 
alcohol.  Distill  the  alcohol  off  from  this  solution,  and  evaporate 
to. a  syrup.  Let  this  stand  until  it  crystallizes.  This  may  be 
hastened  by  the  addition  of  a  little  crystallized  maltose,  which 
can  be  prepared  by  evaporating  a  few  drops  of  the  solution  in  a 
thin  layer  on  a  piece  of  glass.  It  may  be  purified  by  recrystal- 
lizing  from  methyl  alcohol. 

PENTOSES. 

43. — Normally  but  small  amounts  of  the  pentoses  are  found  in 
the  urine  and  these  doubtless  originate  mostly  in  certain  vegetable 
foods.  In  pathological  conditions  they  may  be  more  abundant. 


THE   CARBOHYDRATES. 


Like  glucose,  they  are  reducing  agents,  responding  to  Trommer's 
and  Fehling's  tests.  They  also  form  osazones  which,  however,  have 
different  melting  points  from  the  glucosazone.  They  do  not  fer- 
ment with  yeast,  nevertheless  they  may  be  mistaken  for  reducing 
sugars  because  of  their  other  reactions.  Arabinose,  one  of  the 
most  common,  can  be  obtained  by  boiling  cherry-gum  for  10  to  15 
hours  with  4  per  cent,  sulphuric  acid. 

Prepare  the  arabinose  from  cherry-gum  as  indicated  above, 
neutralize  and  use  for  testing. 

44. — Saturate  5  cubic  centimeters  of  hydrochloric  acid  (30  per 
cent.)  with  phloroglucin  (about  25  milligrammes).  Add  to  this 
the  urine  or  solution  which  is  to  be  tested  for  pentoses  and  heat. 
The  presence  of  pentoses  is  indicated  by  a  red  color  and  this  shows 
absorption  bands  between  D  and  E  of  the  spectrum.  Glucuronic 
acid  gives  a  similar  result. 

45. — Repeat  the  test  as  in  the  last  experiment  substituting 
orcin  for  phloroglucin.  Pentoses  produce  a  green  color.  Glu- 
curonic acid  does  not  act  in  this  manner. 

BEACTIONS  OF  THE  CARBOHYDRATES. 


d 

o 

^"^ 

l*« 

Phenyl- 
hydrazin 

Solubility  in 
Water 

Fermentation 
with  Yeast 

a 
ll 

-2 

s 

a 

Is 
"02 
f, 

5 

r 

Starch     .  .  . 

Blue 

Colloid 
on 

Right 

Dextrins  .... 

Red  to 
Yellow 

•4 

+ 

Heating 

- 

- 

Sweet- 
ish 

- 

Right 

Glycogen  .... 

Red  to 
Violet 

- 

- 

Opal- 
escent 

- 

- 

- 

- 

Right 

Cellulose.  .  .  . 

— 

~ 

— 

— 

— 

— 

— 

— 

.   .   . 

Saccharose    .   . 

- 

- 

- 

+ 

Only  after 
Inversion 

Slight 

Sweet 

+ 

ight 

Maltose 

— 

+ 

+ 

+ 

+ 

Slight 

Sweet 

+ 

Right 

Lactose 

— 

+ 

+ 

+ 

— 

— 

Sweet 

+ 

Kight 

Glucose  .... 

— 

+ 

+ 

+ 

+ 

Slight 

Sweet 

+ 

Right 

Fructose.  .   .  . 

— 

+ 

+ 

+ 

+ 

+ 

Sweet 

+ 

Left 

Arabinose  . 

- 

+ 

+ 

+ 

- 

- 

Sweet 

+ 

Right 

THE  FATS.  23 

THE  FATS. 

The  fats  occur  in  both  plants  and  animals.  When  pure 
they  are  colorless,  odorless,  and  tasteless.  They  are  in- 
soluble in  water  and  have  a  lower  specific  gravity.  They 
dissolve  in  hot  alcohol  more  easily  than  in  cold,  and  are 
easily  soluble  in  ether,  gasolin,  or  benzene.  The  fats  mix 
with  water  when  the  two  are  shaken  violently  together,  but 
they  soon  separate,  the  fats  going  to  the  top.  If,  however, 
something  like  soap  or  a  solution  of  albumin  is  added  to 
the  mixture  which  will  form  a  coating  around  the  minute 
globules  of  fat,  they  are  prevented  from  reuniting,  and 
form  an  emulsion, — that  is,  a  mixture  of  small  fat-globules 
with  the  liquid,  not  a  solution.  It  is  destroyed  by  anything 
which  will  remove  the  coating,  the  fat  separating  again 
from  the  liquid. 

The  fats  are  composed  of  three  elements:  carbon, 
hydrogen,  and  oxygen.  They  contain  a  much  smaller  per- 
centage of  oxygen  than  the  carbohydrates,  the  hydrogen 
and  oxygen  not  being  in  the  proportion  to  form  water. 
When  the  fats  are  kept  at  the  temperature  of  superheated 
steam  or  subjected  to  the  action  of  the  pancreatic  ferment, 
they  take  up  water  and  are  split  into  two  compounds: 
glycerin,  on  the  one  hand,  and  one  or  more  of  the  fatty 
acids,  on  the  other.  Thus,  stearin  gives 

C3H5(C]8H3602)3  +  3H20  =  C3H5(OH)3  +  3C17H35C02H. 

stearin  -f-        water      =  glycerin  +  stearic  acid 

They  may  be  considered  then  as  made  up  of  glycerin 
and  a  fatty  acid  less  water. 

This  splitting  up  of  the  fat-molecule  is  called  saponifi- 
cation.  It  occurs  when  fats  become  rancid.  It  can  be  also 


24  THE   PATS. 

effected  by  boiling  the  fat  with  a  caustic  alkali.  Here,  in- 
stead of  the  free  fatty  acid  being  left,  it  unites  with  the 
alkali  to  form  a  salt.  These  metallic  salts  of  a  fatty  acid 
are  the  soaps: — 

C.HB(C18HMOa)  +  3KOH  =  C3H6(OH)3  +  3C17H85C02K. 

stearin  +          alkali       =  glycerin  +  soap 

The  soaps  of  the  alkalies  are  soluble  in  water,  the 
potassium  compound  being  hygroscopic  and  forming  soft 
soap.  The  sodium  compound  forms  a  hard  soap.  The 
compounds  of  the  heavy  metals  with  the  fatty  acids  are  in- 
soluble, and  can  be  formed  by  adding  a  solution  of  their 
b-alts  to  a  soap  solution.  The  lead  soap  or  lead  plaster  used 
in  medicine  is  made  by  heating  lead  oxid  with  one  of  the 
fats.  The  soluble  soaps  can  be  thrown  down  from  their 
solutions  by  saturation  with  a  neutral  salt.  If  a  strong 
acid  is  added  to  a  soap  solution  the  soap  is  decomposed, 
the  metal  uniting  with  the  strong  acid  and  the  fatty  acid 
being  set  free  as  an  insoluble  substance: — 

C17H3BC02K  +  HC1  =  KC1  +  C17H35C02H. 

goap  acid  fatty  acid  (stearic) 

These  fatty  acids  which  enter  into  the  most  of  the 
animal  and  vegetable  fats  are  one  of  the  unsaturated  series: 
oleic  acid  (C17H33C02H);  and  two  of  the  saturated  series: 
stearic  acid  (C17H35C02H)  and  palmitic  acid  (C15H31C02H). 
Besides  these  acids — which  constitute,  by  far,  the  larger 
part  of  those  present  in  fats — there  are  found  in  some  cases 
certain  of  the  lower  members  of  the  saturated  series,  such 
as  butyric  (C3H7C02H),  capronic  (C^^GO^),  caprylic 
(C7H15C02H),  and  capric  (C9H19C02H),  which  occur  in 
butter. 


THE  PATS.  25 

The  acids  which  enter  into  the  fats  resemble  the  latter 
in  many  of  their  properties.  They  differ  from  them  in 
having  a  slight  acid  reaction,  the  fats  being  neutral.  They 
can  also  be  distinguished  by  their  not  giving  the  irritating 
odor  of  acrolein  as  the  fats  do  when  they  are  heated.  This 
is  produced  by  the  decomposition  of  glycerin,  either  in  a 
fat  or  when  heated  alone.  It  is  most  easily  obtained  by 
adding  before  heating  some  substance  which  will  assist  in 
removing  the  water.  The  chemical  change  is 

C3H5(OH)3  —  2H20  =  C3H40. 

The  free  acids  can  be  neutralized  by  even  weak  alkalies, 
forming  the  soaps. 

The  animal  and  many  of  the  vegetable  oils  are  true  fats, 
differing  only  in  that  they  are  liquids  at  ordinary  tempera- 
tures instead  of  solids.  We  must,  however,  distinguish  be- 
tween these  and  the  essential  or  volatile  oils  which  are 
found  in  plants,  but  which  are  not  fats.  The  fats  produce 
spots  on  paper  which  are  not  volatile  and  do  not  disappear 
on  standing.  The  essential  oils  will  disappear  when  left 
exposed  to  the  air.  The  mineral  oils  belong  to  an  entirely- 
different  class  of  compounds,  and  do  not  contain  oxygen. 

The  fats  are  named  from  the  acid  which  they  contain. 
Thus  the  compound  of  stearic  acid  is  called  stearin;  of 
palmitic  acid,  palmitin;  and  of  oleic  acid,  olein.  Some- 
times the  prefix  tri  is  used  with  these  names,  as  tristearin, 
etc.  They  differ  principally  in  their  melting-points,  olein 
being  a  liquid  at  ordinary  temperatures;  palmitin  and 
stearin,  solids,  the  former  melting  more  easily  than  the 
latter.  In  the  animal  body  these  fats  are  usually  mixed, 
the  consistence  of  the  fat  varying  with  the  composition. 
Thus,  the  fat  of  the  ox,  or  tallow,  is  a  firmer  solid  than 
the  fat  of  the  hog,  or  lard,  because  it  contains  less  of  the 


26  THE   FATS. 

olein.  The  animal  oils  contain  more  olein  than  stearin  and 
palmitin,  consequently  they  melt  at  temperatures  below  the 
ordinary  ones,  and  are  liquids.  Human  fat  contains  67 
per  cent,  to  80  per  cent,  of  olein. 

46. — Try  the  reaction  of  a  fresh  fat,  like  lard  or  olive- 
oil,  with  a  piece  of  litmus-paper.  It  is  neutral ;  but,  if  the 
fat  has  been  standing  some  time  and  has  become  rancid,  it 
may  be  slightly  acid. 

47. — Try  the  solubility  of  a  few  drops  of  olive-oil  in 
a  test-tube  of  water.  It  mixes  when  shaken  violently,  but 
soon  separates  at  the  top  on  standing.  Add  now  a  few 
drops  of  a  soap  solution  and  shake  again.  The  liquid  be- 
comes milky  and  the  fat  does  not  separate.  If  the  oil  is 
not  fresh  it  may  be  necessary  to  add  a  few  drops  of  sodium 
carbonate  to  neutralize  the  free  acid. 

48. — Examine  a  drop  of  the  emulsion  so  formed  under 
the  microscope.  It  will  be  found  to  consist  of  a  great 
number  of  minute  globules  the  size  being  smaller  the  more 
thoroughly  the  liquid  is  shaken.  They  are  kept  apart  by 
the  thin  film  of  soap  which  covers  each  one. 

49. — Add  a  small  amount  of  hydrochloric  acid  and 
shake.  The  soap  will  be  decomposed  and  the  fat  will  col- 
lect at  the  top,  as  at  first. 

50. — Try  the  solubility  of  a  fat  in  ether,  chloroform, 
or  gasolin,  avoiding  carefully  the  vicinity  of  a  flame.  It 
is  easily  soluble. 

51. — Try  the  solubility  of  tallow  or  lard,  first  in  cold,  then  in 
warm,  alcohol.  It  is  readily  soluble  in  the  warm  alcohol  and  sepa- 
rates on  cooling  in  the  crystalline  form.  The  crystals  can  be  ex- 
amined with  the  microscope. 

52. — Show  that  the  fats  are  non-volatile  by -placing 
a  little  upon  paper  and  warming  it  over  a  flame.  It  does 
not  disappear. 


THE  PATS.  27 

53. — To  about  10  grammes  (11  cubic  centimeters)  of 
olive-oil  add  20  cubic  centimeters  of  10-per-cent.  potassium 
hydrate  solution.  Boil  the  mixture,  gently  stirring  mean- 
while, until  the  odor  of  the  oil  has  largely  disappeared  and 
it  appears  homogeneous  and  no  oil  separates  when  a  few 
drops  are  poured  into  water.  This  may  require  fifteen 
minutes  to  half  an  hour.  Add  water  as  it  evaporates,  to 
keep  the  original  volume.  The  product  is  a  mixture  of 
potassium  soap  and  glycerin. 

54. — Convert  a  portion  of  the  soap  into  the  sodium 
or  hard  soap  by  adding  some  saturated  salt  solution  and 
allowing  it  to  stand  until  cold.  It  will  dissolve  on  warm- 
ing. 

55. — To  another  portion  add  a  calcium  solution.  A 
calcium  soap  is  formed  which  is  insoluble  in  water.  It  is 
this  compound  which  is  produced  by  the  action  of  soap  on 
"hard  water."  Many  of  the  heavy  metals  give  similar 
compounds.  Try  it  with  solutions  of  iron,  lead,  copper,  etc. 

56. — To  the  remainder  of  the  potassium  soap  solution 
add  sulphuric  acid  slowly  until  it  is  plainly  acid  to  test- 
paper.  The  fatty  acids  are  set  free  as  insoluble  substances, 
the  glycerin  remaining  in  solution.  Filter  out  the  acids 
by  means  of  a  wet  filter-paper,  through  which  they  will 
not  pass.  Save  the  filtrate  for  the  extraction  of  the  glyc- 
erin. Wash  out  the  sulphuric  acid  with  distilled  water 
until  the  wash-water  is  no  longer  acid,  and  try  the  reaction 
of  the  fatty  acids  with  litmus-paper.  They  are  acid  to 
litmus. 

57. — Dissolve  the  fatty  acids  in  hot  alcohol,  let  this 
cool  slowly,  observe  and  sketch  the  crystals. 

58. — Allow  the  fatty  acids  to  stand  until  the  water  has 
drained  off  or  dry  them  by  the  aid  of  filter-paper.  Heat  them  in 


28  THE  FATS. 

a  dry  test-tube  until  they  commence  to  decompose,  and  see  if  they 
give  the  irritating  odor  of  acrolein.  If  the  acids  were  washed 
clean  and  if  the  fat  was  completely  saponified  this  should  not  ap- 
pear. Try  the  same  test  on  any  of  the  fats,  and  the  odor  will  be 
given.  It  is  best  produced  by  adding  before  heating  a  little  acid 
potassium  sulphate. 

59. — Neutralize  the  first  filtrate  from  the  acids,  which 
contains  the  glycerin,  by  adding  a  little  powdered  chalk, 
then  evaporate  the  mixture  to  complete  dryness  on  a  water- 
bath.  Extract  the  glycerin  from  the  powdered  mass  by 
alcohol  and  evaporate  this  on  the  water-bath.  If  the  glyc- 
erin is  to  be  further  purified  this  residue  should  be  ex- 
tracted with  absolute  alcohol  and  the  alcoholic  filtrate 
evaporated.  The  thick  liquid  is  the  glycerin,  as  is  shown 
by  the  taste. 

60. — Mix  2  grammes  of  lead  monoxid  (litharge)  with  11  cubic 
centimeters  of  olive-oil  and  75  cubic  centimeters  of  water.  Boil 
until  the  oil  is  entirely  converted  into  the  lead  soap.  This  is 
"lead  plaster."  Filter  it  off;  wash  the  insoluble  soap  with  water 
and  evaporate  the  filtrate  with  the  wash-water  in  an  evaporating 
dish  on  a  waterbath,  as  long  as  its  volume  decreases.  The  glycer- 
in remains.  It  contains  lead;  if  the  pure  substance  is  desired 
this  can  be  removed  by  diluting  with  water  and  passing  hydrogen 
sulphid  into<  it. 

Glycerin  dissolves  many  metallic  oxids,  e.g.,  precipitated, 
washed  cupric  oxid  dissolves  to  a  blue  liquid.  Try  this.  A  borax 
bead  when  dipped  in  glycerin  gives  a  green  color  when  heated  in 
the  Bunsen  flame* 

61.  THE  PREPARATION  OF  PURE  PALMITIC  ACID. — Melt  in 
hot  water  10  grammes  of  bayberry  wax  which  is  nearly  pure 
palmitin  and  add  20  cubic  centimeters  of  a  20  per  cent,  solution  of 
sodium  hydrate.  Boil  it  until  a  drop  makes  a  soapy  solution 
when  poured  into  a  test-tube  of  water.  Sodium  palmitate  has 
been  formed  together  with  glycerin,  carbon  dioxid  and  water. 
(Write  the  equation  for  the  reaction.)  Acidify  slightly  the  soap 


THE   FATS.  29 

solution  with  dilute  hydrochloric  acid,  filter  out  the  palmitic  acid, 
wash  it  with  cold  alcohol  until  it  is  white,  dissolve  it  in  the  least 
possible  quantity  of  hot  alcohol  and  let  it  stand  until  it  is  cold. 
Examine  with  a  microscope  the  crystals  which  have  separated  and 
make  sketches  of  them.  Test  the  reaction  to  blue  litmus  paper. 

62.  THE  PREPARATION  OF  PURE  SOAP. — Melt  at  a  low  tem- 
perature most  of  the  palmitic  acid  from  the  last  experiment  and 
slowly  add  sodium  hydrate  solution  until  the  reaction  is  alkaline, 
warming  meanwhile.  A  hard  soap  is  produced.  (Write  the  equa- 
tion.) If  a  little  alcohol  is  mixed  with  this  before  it  cools  a 
transparent  soap  results. 

63. — Hold  a  piece  of  small  glass  tubing  in  the  Bunsen  flame 
until  it  is  soft,  turning  it  continually.  Then  draw  it  out  into  a 
capillary  tube  about  six  inches  long.  Break  this  in  the  middle, 
forming  two  open  capillaries  each  with  a  large  end.  Melt  a  fat 
at  low  temperature  and  draw  it  up  into  the  capillary  by  exhaust- 
ing the  air,  allowing  it  to  cool  and  solidify  there.  Seal  up  the 
large  opening  by  means  of  a  blast  lamp  or  Bunsen  burner.  At- 
tach the  tube  to  the  bulb  of  a  thermometer  by  means  of  a  rubber 
band  cut  from  a  piece  of  tubing.  Hold  this  in  a  beaker  of  water, 
warming  the  latter  slowly.  Make  a  note  of  the  melting  point — 
the  temperature  at  which  the  fat  liquifies.  In  this  manner  deter- 
mine the  melting  points  of  a  number  of  fats,  like  mutton-tallow, 
beef-tallow,  lard,  butter,  also  of  palmitic  acid  and  stearic  acid, 
beef  tallow,  lard,  butter,  also  of  palmitic  acid  and  stearic  acid. 
Hand  in  the  results.  Place  the  thermometer  with  the  capillary 
of  melted  fat  in  water  a  little  above  its  melting  point  and  let  it 
cool  slowly,  noting  the  point  at  which  solidification  occurs.  Notice 
that  this  is  below  the  melting  temperature  and  that  it  varies  with 
the  speed  of  cooling. 

64. — Try  the  solubility  of  fate,  fatty  acids  and  soaps  in 
chloroform,  ether,  water,  hot  and  cold  alcohol.  Tabulate  the  re- 
sults and  determine  what  methods  can  be  employed  for  their 
separation. 

65. — Make  the  acrolein  test  upon  the  water-free  glycerin  by 
heating  in  a  dry  tube  with  some  crystals  of  potassium  bisulphate. 
The  glycerin  is  decomposed,  giving  the  acrolein  odor,  and  showing 
that  the  odor  when  obtained  from  fats  is  due  to  the  glycerin  radi- 
cal, and  not  to  the  acid  part. 


30  THE  LECITHINS. 

66.  —  Show  that  olive-oil  is  not  a  simple  fat,  but  a 
mixture,  by  cooling  it  to  the  freezing-point  either  by  nat- 
ural cold  or  by  a  freezing  mixture.  It  separates  into  two 
parts:  one  crystalline,  consisting  mostly  of  palmitin,  and 
the  other  olein,  which  remains  liquid. 

THE  LECITHINS. 

The  lecithins  are  found  in  nearly  all  animal  and  vege- 
table cells.  They  are  very  abundant  in  the  brain  and 
nerves  and  in  the  yelk  of  eggs.  They  are  sometimes  called 
phosphorized  fats.  The  formula  of  one  of  those  most  com- 
mon in  the  animal  body  is.C44H90KP09,  and  the  composi- 
tion is  probably 


CH0 


It  is  therefore  a  fat  where  for  one  stearic  acid  radical 
has  been  substituted  the  group 


-P03OH  C2H4  N(CH3)3  OH. 


In  others,  instead  of  the  stearic  acid  radical  we  may 
have  the  oleic  or  palmitic  radical.  Like  other  fats,  they 
can  be  decomposed  or  saponified.  They  then  give  phos- 
phoric acid,  a  fatty  acid,  and  a  base,  cholin: — 

HO  C2H4  N(CH3)3  OH. 

Lecithin  is  a  soft,  waxy  substance,  which  swells  in 
water  to  a  pasty  mass.  This,  under  the  microscope,  has  the 
form  of  oily  drops  or  threads,  the  so-called  "myelin"  forms. 
It  resembles  nuclein  in  the  readiness  with  which  it  unites 


THE   LECITHINS.  31 

with,  albuminous  substances.    It  is  found  in  the  yelk  of  the 
egg  in  an  unstable  union  with  vitellin. 

67.  PREPARATION  OF  LECITHIN. — In  the  following  work  the 
student  should  remember  that  ether  and  petroleum-ether  are  very 
inflammable. 

Separate  the  albumin  of  an  egg  as  completely  as  possible  from 
the  yelk.  Place  the  yelk  in  a  cylindrical,  glass-stoppered  bottle, 
add  two  or  three  times  its  volume  of  ether,  and  shake  the  bottle 
until  they  are  well  mixed.  Allow  it  to  stand  until  the  ether  above 
becomes  clear,  and  then  decant  the  latter  into  a  distilling  flask.  Re- 
peat this  extraction  several  times,  when  most  of  the  coloring  matter 
should  have  been  dissolved.  Preserve  the  insoluble  for  the 
preparation  of  hsematogen.  Mix  the  portions  of  ether  and  distill 
off  the  ether.  The  residue  contains  the  lecithin  mixed  with  fats, 
cholesterin,  and  coloring  matters.  Dissolve  this  in  petroleum- 
ether  and  filter.  Pour  the  filtrate  into  a  separatory  funnel,  add 
about  one-fourth  its  volume  of  75-per-cent.  alcohol,  and  shake. 
When  the  two  liquids  have  separated  draw  off  the  alcohol,  which 
contains  most  of  the  lecithin.  Repeat  this  extraction  with  alcohol 
several  times  and  unite  the  alcoholic  solutions.  Distill  off  the 
remainder  of  the  petroleum-ether  from  these,  and  let  the  solution 
stand  several  days  in  a  cool  place.  A  precipitate  of  cholesterin 
and  other  impurities,  will  form,  from  which  the  solution  is  to  be 
decanted  through  a  filter.  Boil  the  filtrate  with  a  little  animal 
charcoal  to  decolorize  it,  and  filter.  Evaporate  at  a  temperature 
of  50°  to  60°  to  a  syrupy  consistency.  Cool  this  and  dissolve  in 
ether.  If  it  does  not  dissolve  completely,  filter  it.  Evaporate  the 
ether,  when  the  lecithin  remains  nearly  pure.  If  desired  it  can 
be  purified  further  by  dissolving  in  as  small  an  amount  as  possible 
of  warm  absolute  alcohol  and  placing  this  in  a  freezing  mixture 
of  — 5°  to  — 159,  when  the  lecithin  crystallizes  out.  It  should  be 
filtered  in  the  cold. 

68. — Place  a  little  lecithin  in  water  and  examine  with  the  mi- 
croscope. Notice  the  myelin  forms. 

69. — Warm  this  mixture  with  water  and  notice  that  after  a 
time  the  lecithin  turns  brown  and  the  reaction  becomes  acid  from 
decompo  sition. 


32  THE   PBOTEINS. 

70. — Mix:  a  little  lecithin  with  dry,  powdered  potassium  nitrate 
in  a  small  porcelain  crucible,  and  warm,  at  first  gently,  then,  after 
deflagration,  until  the  dark  color  has  disappeared.  After  cooling 
dissolve  in  water  and  test  for  phosphoric  acid  by  nitric  acid  and 
ammonium  molybdate.  At  once  or  after  warming  a  yellow  pre- 
cipitate will  appear. 

THE  PKOTEINS. 

The  protein  compounds  constitute  the  greater  part  of 
the  solid  matter  of  the  blood,  muscles,  nerves,  and  other 
organs  of  the  animal  hody.  The  urine,  tears,  and  perspira- 
tion, in  a  normal  condition,  never  contain  more  than  a 
trace.  The  proteins  contain  carbon,  hydrogen,  nitrogen, 
oxygen,  and  usually  sulphur.  A  few  contain  phosphorus 
and  a  few  others  iron.  When  heated  they  are  charred, 
giving  off  water,  inflammable  gases,  and  ammonia,  at  the 
same  time  emitting  a  strong  odor  similar  to  that  of  burnt 
horn  or  wool.  Upon  further  ignition  they  leave  an  ash, 
though  whether  this  was  originally  a  part  of  the  protein 
molecule  has  not  been  decided.  They  are  often  spoken  of 
simply  as  the  nitrogenous  constituents  of  the  body  or  the 
food,  although  not  all  of  the  nitrogenous  compounds  found 
there  belong  to  this  class. 

The  proteins  are  very  complex  substances  with  a  high 
molecular  weight,  and  it  is  probably  owing  to  this  fact 
that  they  are  so  easily  decomposed,  as  is  seen  by  the  putre- 
faction which  sets  in  soon  after  life  has  ceased.  To  the 
large  molecule,  too,  is  probably  due  the  inability  of  most 
of  them  to  pass  through  a  parchment  or  animal  membrane. 

GENERAL  PROPERTIES  OF  THE  PROTEINS. 

71. — Burn  a  small  piece  of  dry  albumin  or  other 
protein  compound  on  a  piece  of  porcelain  or  platinum  foil, 


PROPERTIES   OF   THE  PROTEINS.  33 

or  on  a  wire.  Notice  that  it  turns  black  from  the  presence 
of  carbon.  Observe  the  characteristic  odor.  On  continued 
heating  it  will  all  disappear  except  the  mineral  matters, 
or  ash. 

72. — Mix  a  few  fragments  of  dry  albumin  with  an 
excess  of  powdered  soda-lime  and  heat  the  mixture  in  a 
dry  test-tube.  Test  the  vapors  which  escape  for  ammonia, 
both  by  the  odor  and  by  their  action,  on  a  piece  of  red  lit- 
mus-paper. The  ammonia  proves  that  the  albumin  con- 
tained nitrogen. 

73. — Make  a  solution  of  lead  hydrate  by  adding  so- 
dium hydrate  to  a  small  amount  of  lead  acetate  solution 
until  the  precipitate  first  formed  has  dissolved.  Add  to  it 
a  protein  compound,  like  albumin,  and  boil.  The  presence 
of  sulphur  (cystein  sulphur)  in  the  protein  compound  is 
shown  by  the  dark-colored  lead  sulphid,  which  it  forms  by 
uniting  with  the  lead. 

74. — Very  small  quantities  of  sulphur  maybe  shown  by  chang- 
ing it  to  a  sulphid  and  testing  for  the  latter  with  sodium  nitro- 
prussid.  By  this  means  it  can  be  found  in  a  single  hair.  Shut  off 
the  air  from  a  Bunsen  burner  and,  after  turning  it  low,  cover  the 
hair  with  sodium  carbonate  and  hold  it  on  a  wire  in  the  middle  of 
the  flame.  Allow  the  substance  to  fuse,  being  careful  to  keep  it  in 
the  yellow  flame  to  prevent  oxidation.  Then  dissolve  the  mass  in 
a  few  drops  of  water  in  a  porcelain  dish.  Add  to  the  solution  a 
very  small  crystal  of  sodium  nitroprussid.  The  presence  of  sul- 
phid is  shown  by  the  production  of  a  purple  or  violet  color,  which 
is  destroyed  by  an  excess  of  the  nitroprussid. 

75. — Test  a  solution  of  egg-albumin  or  any  other  albuminous 
substance  to  see  if  it  will  pass  through  a  dialyzer.  Only  the  pep- 
tones will  pass  through  the  membrane.  The  biuret  test  can  be 
used  to  detect  them  (Experiment  76). 


34 


THE   PROTEINS. 


CLASSIFICATION  OF  THE  PRINCIPAL  PROTEIN  COMPOUNDS. 


Albumin. 

Globulin. 

Albuminous 
substances 

Albuminates  -j 

Acid  albumin. 
Alkali  albumin. 

or  simple 

Proteoses. 

proteins 

Peptones. 

Fibrin. 

/">  ,,1«J.«J     „!! 

Proteins 


Compound 
proteins 


I  Albuminoids  - 


Coagulated  albumin. 

Mucin. 
Hemoglobin. 
Nucleoalbumin. 
Nuclein. 

f  Keratin. 

Elastin. 

Collagen. 

Gelatin. 
I  Etc. 


ALBUMINOUS  SUBSTANCES. 

These  are  sometimes  called  proteins,  though  it  is 
more  convenient  to  reserve  this  name  for  the  whole  class, 
including  also  the  albuminoids.  They  form  the  principal 
part  of  the  protoplasm  which  is  found  in  animal  and  plant 
cells.  The  constitution  of  the  molecule  and  even  the 
exact  formula  of  the  different  members  of  the  group  is 
uncertain.  They  are  known  to  be  very  complex,  those 
which  have  been  most  studied  having  several  hundred 
atoms'  in  a  molecule.  They  differ  somewhat  from  each 
other  in  composition,  but  their  constituents  usually  lie 
within  the  following  limits; — 


substance. 


ALBUMINOUS   SUBSTANCES.  35 

Average  of  Most  Analyses.  Approximation. 

C  50.0  to  55.0  per  cent.  52  per  cent. 

H  6.5  to    7.3  per  cent.  7  per  cent. 

0  20.0  to  23.5  per  cent.  23  per  cent. 

S  0.3  to    2.2  per  cent.  2  per  cent. 

N  15.0  to  18.0  per  cent.  16  per  cent. 

Phosphorus  is  sometimes  found  in  less  amounts  than 
1  per  cent. 

A  few  of  the  albuminous  substances  have  been  ob- 
tained in  a  crystalline  form,  but  most  of  them  are  amor- 
phous. They  differ  in  their  solubilities  and  are  classified 
largely  upon  this  basis.  The  peptones  will  diffuse  through 
an  animal  membrane,  but  they  do  not  pass  through  rapidly. 

The  albuminous  substances,  like  some  other  organic 
compounds  which  do  not  belong  to  this  class,  are  thrown 
out  of  solution  when  to  the  solution  certain  neutral  salts 
are  added  until  it  is  saturated.  Ammonium  sulphate  will 
precipitate  all  but  the  peptones  and  perhaps  a  few  of  the 
albumoses.  Magnesium  sulphate  and  sodium  chlorid  will 
precipitate  many  of  them. 

When  the  albuminous  substances  are  heated  with 
water,  many  of  them  are  coagulated,  passing  into  an  in- 
soluble modification.  The  temperature  at  which  this  takes 
place  is  called  the  coagulation-point.  This  is  a  different 
one  for  most  of  the  different  substances,  and  may  be  used 
in  their  identification  and  separation.  It  may  vary,  how- 
ever, from  the  presence  of  other  substances.  It  may  be 
raised,  prevented,  or  the  coagulation  made  incomplete  by 
alkalies  or  by  some  organic  acids,  like  acetic  acid.  Coagu- 
lation is  favored  and  the  coagulation-point  is  lowered  in 
the  presence  of  neutral  salts  or  small  amounts  of  a  mineral 


36  THE   PROTEINS. 

acid.  The  concentration  of  the  solution  also  may  make 
it  vary.  Through  coagulation  the  nature  of  albuminous 
substances  is  altered  and  they  acquire  other  properties.  By 
the  action  of  alcohol  albuminous  compounds  are  precipi- 
tated, at  first  in  an  unaltered  form;  but  if  the  alcohol  is 
strong  and  acts  for  some  time  they  are  coagulated,  and  are 
then  insoluble  in  water. 

Coagulation,  when  spoken  of  with  respect  to  the 
protein  compounds,  must  be  distinguished  from  precipita- 
tion, which  it  resembles.  When  albumin  is  coagulated — 
e.g.,  by  boiling,  by  mineral  acids,  or  by  the  continued 
action  of  strong  alcohol — it  becomes  insoluble  in  water. 
It  may  be  precipitated  by  ammonium  sulphate  without  be- 
ing coagulated  or  by  not  too  large  an  amount  of  alcohol 
and  still  retain  its  original  properties,  being  soluble  again 
upon  the  addition  of  water. 

Some  of  the  albuminous  compounds  are  coagulated  by 
the  action  of  ferments;  for  example,  the  fibrin,  which  is 
so  formed  from  the  blood  or  lymph. 

Albuminous  substances  are  easily  decomposed  by  the 
action  of  the  putrefactive  bacteria,  the  nitrogen  and  sul- 
phur uniting  with  hydrogen  to  form  hydrogen  sulphid, 
and  ammonia,  or,  these  two  together,  ammonium  sulphid. 
Other  nitrogen  compounds  are  also  formed,  like  the  amido 
acids  which  contain  the  amido  group,  KH2,  such  as  leucin 
and  tyrosin.  Indol  is  also  one  of  the  nitrogenous  putre- 
factive products. 

Many  of  the  albuminous  substances  are  precipitated 
by  the  mineral  acids,  but  upon  standing  with  an  excess  of 
the  acid,  or  more  quickly  by  heating,  they  are  dissolved, 
going  into  acid  albumins.  Many  will  also  form  insoluble 
compounds  with  salts  of  the  heavy  metals,  such  as  mercury, 
copper,  and  lead.  With  copper  in  an  alkaline  solution  they 


REACTIONS   OF  ALBUMINOUS   SUBSTANCES.  37 

give  a  blue  or  purple  color  and  upon  boiling  with  an  excess 
of  nitric  acid  a  yellow,  which  becomes  more  reddish  upon 
being  rendered  alkaline.  Millon's  reagent,  which  gives  a 
red  with  all  compounds  containing  a  benzene  nucleus 
united  with  an  hydroxyl  group,  produces  the  same  color 
with  albuminous  compounds,  whence  it  is  believed  that 
the  above  complex  is  contained  in  albumins.  The  xan- 
thoproteic  reaction  is  attributed  to  the  same  or  the  indol 
group.  Similarly  each  of  the  other  tests  appears  to  be 
produced  by  some  definite  constituent  of  the  protein  mole- 
cule. 

GENERAL  REACTIONS  OF  THE  ALBUMINOUS  SUBSTANCES. 

The  tests  may  be  made  upon  any  albuminous  com- 
pound ;  for.  example,  egg-albumin. 

76. — Make  the  solution  alkaline  with  sodium  hydrate 
and  add  a  few  drops  of  a  dilute  cupric  sulphate  solution. 
A  blue  or  purple  color  results.  An  excess  of  the  copper 
solution  must  be  carefully  avoided,  as  it  may  produce  a 
blue  color  when  no  protein  compound  is  present.  (Biuret 
test.) 

77. — Add  a  small  quantity  of  concentrated  nitric  acid 
to  the  albumin  solution  and  heat  to  boiling.  A  yellow  color 
is  produced  which  becomes  orange  red  when  the  liquid  is 
made  alkaline  with  sodium  hydrate  or  ammonia.  (Xantho- 
proteic  reaction.) 

78. — Make  the  solution  of  albumin  acid  with  acetic 
acid,  then  add  at  least  an  equal  volume  of  a  saturated  solu- 
tion of  ammonium  sulphate,  and  heat  to  boiling.  Most 
albuminous  compounds  are  thrown  down  as  a  white  pre- 
cipitate. 


38 


THE   PROTEINS. 


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ALBUMINS.  39 

79. — Acidify  the  solution  with  acetic  acid  and  add  a 
few  drops  of  potassium  ferrocyanid.  A  white  precipitate  is 
formed. 

The  two  last  reagents  fail  in  case  of  the  peptones. 

80. — Add  an  excess  of  Millon's  reagent  and  boil.  A 
red  color  is  produced  with  all  albuminous  compounds  ex- 
cept antipeptones. 

81. — Many  of  the  albuminous  compounds  are  also  precipi- 
tated by,  (1)  trichloracetic  acid;  (2)  metaphosphoric  acid;  (3) 
alcohol  or  ether;  (4)  from  solutions  acidified  with  HC1  or  HNO3 
by  potassium  mercuric  iodid  or  phosphotungstic  acid;  (5)  from 
acetic  acid  solutions  by  picric  or  tannic  acid;  (6)  from  weakly 
alkaline  solutions  by  salts  of  mercury  and  some  of  the  other  heavy 
metals.  Try  these. 

No  reaction  is  characteristic  in  itself;  therefore  in  test- 
ing for  these  bodies  a  number  of  tests  should  ~be  tried. 

ALBUMINS. 

The  albumins  are  soluble  in  water.  They  are  not  pre- 
cipitated by  dilute  acids  or  alkalies.  They  are  precipitated 
by  saturation  with  ammonium  sulphate  and  by  some  of  the 
salts  of  the  heavy  metals,  such  as  mercuric  chlorid  and 
salts  of  copper,  silver,  and  lead.  For  this  reason  albumin 
is  successfully  used  as  an  antidote  for  poisoning  by  many 
of  these  metallic  salts.  By  the  action  of  acids  it  is  con- 
verted into  acid  albumin  and  by  the  caustic  alkalies  into 
alkali  albumin. 

There  are  several  different  varieties  of  albumin  usually 
named  from  their  sources,  such  as  the  serum-albumin  of 
the  blood;  egg-albumin;  and  the  albumin  of  milk,  or 
lactalbumin.  We  know  that  these  are  not  the  same  sub- 
stances from  their  physiological  action,  as  well  as  from 
some  chemical  and  physical  properties.  If,  for  example, 


40  THE   PROTEINS. 

egg-albumin  be  injected  into  the  circulation  it  usually 
passes  in  a  short  time  into  the  urine.  Serum-albumin  in- 
jected in  the  same  manner  does  not  pass  unaltered  through 
the  kidneys.  Serum-albumin  is  more  easily  dissolved  by 
an  excess  of  mineral  acid  than  egg-albumin.  They  differ 
also  in  their  coagulation-temperatures. 

Albumin  may  be  obtained  for  the  experiments  by  dis- 
solving the  commercial  dry  egg-albumin  or  serum-albumin 
in  cold  water,  filtering  if  it  is  not  clear.  From  fresh  eggs 
it  can  be  prepared  by  separating  the  albumin  from  the 
yelk,  being  careful  not  to  mix  them.  Then  beat  the  albu- 
min, to  break  up  the  membranes  in  it,  mix  with  twice  its 
volume  of  water,  and  filter  through  a  piece  of  unbleached 
muslin. 

82. — Place  a  moistened  piece  of  parchment  or  a  parch- 
ment filter  in  a  funnel  from  which  the  stem  has  been 
broken,  and  hang  the  funnel  in  a  beaker  of  water  so  that 
the  water  rises  nearly  to  the  top.  Into  the  parchment  pour 
a  solution  of  egg  or  serum  albumin  with  a  little  salt,  and 
let  it  stand  all  night.  Test  the  outer  liquid  by  the  xantho- 
proteic  and  biuret  reactions  for  proteids.  None  will  be 
found,  although  it  contains  a  chlorid,  as  shown  by  adding 
silver  nitrate  with  nitric  acid,  when  a  white  curdy  precipi- 
tate of  silver  chlorid  appears. 

83. — Tie  a  piece  of  moistened  parchment  over  the 
mouth  of  a  thistle  tube,  then  fill  the  bulb  with  an  albumin 
solution  and  suspend  the  tube  by  a  clamp,  so  that  the  bulb 
is  covered  by  the  water  in  a  beaker  below.  The  liquid  rises 
in  the  tube  above  the  level  of  that  outside  owing  to  the 
inward  osmotic  pressure  of  the  water  being  greater  than 
the  outward  one  of  the  albumin  solution.  This  is  the 
action  that  occurs  when  animal  cells  are  surrounded  by 
pure  water. 


ALBUMIN.  41 

Purified  egg-albumin  may  be  prepared  by  the  following 
method: — 

84.— Separate  the  white  from  the  yelk  and  cut  it  for  some 
time  with  scissors  or  beat  it  up  to  a  strong  foam.  Allow  this  to 
subside;  filter  or  press  it  through  clean,  unsized  muslin;  mix  it 
with  an  equal  volume  of  water,  and,  after  the  precipitate  has  sub- 
sided, filter  it.  Saturate  the  filtrate  at  about  20°  with  very  finely 
powdered  magnesium  sulphate,  added  in  small  portions  with  con- 
tinual stirring.  When  it  is  saturated  filter  off  the  precipitated 
globulins  and  dialyze  the  filtrate  until  a  portion  gives  only  a  faint 
turbidity  with  barium  chlorid.  The  solution  will  increase  in  vol- 
ume during  the  operation.  It  should  be  concentrated  in  flat  dishes 
at  40°  to  50°,  again  dialyzed,  and  finally  evaporated  to  dryness  at 
the  above  temperature.  It  still  contains  1  to  3  per  cent,  of  ash. 

85. — To  obtain  purified  serum-albumin  the  blood  must  be 
drawn  into  a  dry  vessel  and  the  fibrin  coagulated  by  beating. 
Filter  this  out  through  muslin  and  separate  the  corpuscles  from 
the  serum  by  a  centrifugal  machine  or  by  allowing  it  to  stand  in  a 
tall  cylinder  until  the  corpuscles  have  settled.  Horse-blood,  if 
obtainable,  is  best  for  this  purpose.  Then  draw  off  the  clear 
serum  with  a  siphon,  dilute  it  with  three  volumes  of  a  saturated 
solution  of  ammonium  sulphate,  and  add  gradually,  stirring  con- 
tinually, finely  powdered  ammonium  sulphate  until  the  liquid  is 
saturated.  Filter  off  the  precipitate,  which  contains  serum- 
albumin  and  paraglobulin,  and  wash  it  with  a  saturated  solution 
of  ammonium  sulphate.  It  will  be  purer  if  it  is  dissolved  in  a 
small  quantity  of  water  and  the  precipitating  and  washing  are 
repeated.  Dissolve  the  precipitate  while  it  is  still  moist  in  the 
smallest  possible  quantity  of  water  and  dialyze  it.  The  para- 
globulin  separates  out  as  a  white  flocculent  precipitate,  while  the 
albumin  remains  in  solution.  Filter  out  the  globulin  and  wash 
it  with  water.  It  is  insoluble  in  water,  but  soluble  in  dilute 
solutions  of  neutral  salts.  It  is  precipitated  by  neutral  salts  like 
ammonium  sulphate,  magnesium  sulphate,  or  sodium  chlorid.  Its 
sodium  chlorid  solution  coagulates  at  about  75°. 

If  the  solution  of  serum-albumin  contains  no  globulin,  it  will 
not  be  made  turbid  by  adding  to  a  small  portion  magnesium  sul- 
phate to  saturation.  In  that  case  it  is  to  be  carefully  neutralized 
with  ammonia,  the  salts  dialyzed  out,  the  contents  of  the  dialyzer 


42  THE   PROTEINS. 

filtered,  if  necessary,  and  evaporated  at  40°  to  a  small  volume. 
The  solution  should  be  precipitated  by  alcohol  and  immediately 
filtered.  Press  out  the  excess  of  alcohol  as  much  as  possible,  and 
wash  out  the  remainder  with  ether.1  Allow  the  ether  to  escape  by 
stirring  the  albumin  in  a  mortar. 

86.  THE  PREPARATION  OF  A  CRYSTALLIZED  VEGE- 
TABLE PROTEIN. — The  edestin  of  the  hemp  seed  will  serve 
as  an  example.  Digest  about  10  grammes  of  ground  hemp 
seed  for  half  an  hour  at  50°  to  60°  with  about  50  cubic 
centimeters  of  10  per  cent,  sodium  chlorid  solution;  then 
filter.  Divide  the  nitrate  into  two  parts.  Dilute  one  part 
with  warm  water  until  it  begins  to  be  turbid.  Let  it  cool 
very  slowly  to  nearly  the  freezing  point.  Put  the  second 
part  in  a  dialyzer  and  allow  the  salt  to  diffuse  into  the 
surrounding  water.  In  both  cases  microscopic  crystals 
will  be  found,,  octahedral  or  tetrahedral  in  form.  The 
more  slowly  they  are  formed  the  more  perfect  they  will  be. 

87. — Show  that  the  crystals  are  insoluble  in  water, 
but  soluble  in  salt  solutions.  This  solution  precipitates 
on  dilution  with  water  and  coagulates  by  heating.  It  also 
gives  the  Millon's  and  the  xanthoproteid  reactions.  The 
edestin  is  therefore  a  globulin. 

88.  THE  PREPARATION  OF  CRYSTALLIZED  EGG  ALBUMIN.— 
To  100  cubic  centimeters  of  egg-white  from  fresh  eggs  add  an 
equal  volume  of  a  saturated  solution  of  ammonium  sulphate. 
Beat  the  mixture  thoroughly  with  an  egg-beater  or  a  fork  to  break 
up  the  membranes  and  let  it  stand  in  a  cool  place  for  several 
hours,  then  filter  through  muslin  or  paper  until  the  filtrate  is  clear. 
Add  to  the  filtrate  more  saturated  ammonium  sulphate  until  a 
permanent  precipitate  forms,  after  which  drop  in  distilled  water 
until  this  dissolves,  avoiding  an  excess.  Now  add  acetic  acid, 


1This  must  be  done  away  from  the  vicinity  of  any  light  or 
fire. 


ALBUMIN.  43 

drop  by  drop,  until  a  slight  precipitate  appears,  and  let  it  stand 
over  night,  when  there  should  be  a  separation  of  the  albumin  in 
rosette-like  clusters  of  needle-shaped  crystals.  If  the  eggs  are 
not  fresh  a  little  more  acid  must  be  used — enough  to  make  a  fairly 
bulky  precipitate — and  then  the  solution  must  stand  over  night  in 
a  closed  flask.  To  recrystallize,  dissolve  in  cold  water,  add  a  few 
drops  of  acetic  acid,  then  saturated  ammonium  sulphate  solution 
as  before  till  the  precipitate  commences  to  form  and  let  it  stand. 
The  yield  should  be  about  40  per  cent. 

89. — Try  the  effect  of  various  reagents  upon  the  co- 
agulation-temperature of  albumin  by  placing  about  10 
cubic  centimeters  of  an  albumin  solution  in  a  number  of 
test-tubes,  first  filtering  if  necessary  to  obtain  a  clear 
liquid,  and  adding  to  each  three  drops  of  the  following 
solutions  (1)  sodium  chlorid;  (2)  concentrated  sodium 
chlorid  and  acetic  acid;  (3)  dilute  hydrochloric  acid; 
(4)  sodium  hydrate;  (5)  a  solution  of  sodium  carbonate; 
(6)  nothing.  Set  the  test-tubes  in  a  beaker  of  water  and 
heat  to  boiling,  observing  which  coagulate  first.  It  will  be 
found  that  acids  and  neutral  salts  favor  coagulation  and 
that  alkalies  hinder  it  or  prevent  it  altogether. 

90. — Eather  strongly  acidify  a  concentrated  solution 
of  albumin  with  hydrochloric  or  sulphuric  acid.  The  albu- 
min is  precipitated.  Is  it  coagulated?  Test  by  diluting 
with  water. 

91. — Suspend  a  small  beaker  in  a  larger  one,  keeping  them 
separate  by  wedges  of  cork;  fasten  by  a  clamp.  Nearly  fill  both 
with  water  and  place  in  the  inner  one  a  test-tube  with  enough 
solution  of  filtered  egg  albumin  to  stand  at  the  same  level  as  the 
water.  The  test-tube  should  not  touch  the  beaker  wall.  Heat 
the  water  gradually,  having  a  thermometer  in  the  albumin  solu- 
tion. Above  45°  the  temperature  should  not  be  allowed  to  rise 
faster  than  1°  per  minute.  Make  a  note  of  the  temperature  where 
the  liquid  becomes  opalescent  and  where  a  precipitate  forms. 


44  THE   PHOTEINS. 

92. — Put  some  of  the  clear  albumin  solution  into  three  test- 
tubes.  Make  one  of  them  faintly  acid  by  a  drop  of  very  dilute 
hydrochloric  acid;  add  to  the  second  enough  of  a  very  dilute  solu- 
tion of  sodium  carbonate  to  make  it  faintly  alkaline;  have  the 
third  exactly  neutral.  Heat  them  as  in  the  last  experiment,  ob- 
serving the  coagulation  temperature  by  means  of  a  sensitive  ther- 
mometer. Report  results. 

93. — Test  the  solubility  in  cold  water  of  some  egg- 
albumin  which  has  been  dried  at  a  low  temperature.  It 
dissolves  slowly,  showing  that  coagulation  has  not  occurred. 
The  solution  will  give  the  albumin  reactions. 

94. — Place  some  of  this  thoroughly  dry  egg-albumin 
in  a  dry  test-tube  and  let  this  stand  for  several  minutes 
in  a  beaker  of  boiling  water.  After  removal  it  will  dis- 
solve in  cold  water  as  before.  Coagulation  has  not  taken 
place,  since  for  this  the  presence  of  water  is  necessary. 

95. — Test  solutions  of  serum-albumin  and  egg-albu- 
min with  an  excess  of  nitric  acid  without  heat  and  also  by 
warming,  and  notice  that  the  serum-albumin  is  more 
easily  soluble.  Try  the  same  with  concentrated  hydro- 
chloric acid. 

96. — Show  that  a  solution  of  egg-albumin  forms  an 
insoluble  compound  when  added  to  solutions  of  mercury, 
silver,  or  copper. 

GLOBULINS. 

The  globulins  are  distinguished  from  all  the  other 
albuminous  substances  by  being  soluble  in  dilute  salt  solu- 
tions, but  insoluble  in  water.  From  this  solution  they  are 
precipitated  by  diluting  freely  with  water  or  by  removing 
the  salt  by  diatysis.  They  can,  in  this  manner,  be  sepa- 
rated from  the  albumins  which  remain  in  solution.  The 
globulins  are,  as  a  rule,  precipitated  by  saturating  the 


GLOBULINS.  45 

solution  with  neutral  salts,  like  'sodium  chlorid.  They 
are  coagulated  by  heating  with  water.  By  the  action  of 
dilute  acids  they  are  converted  into  acid  albumin.  A  com- 
mon example  is  myosin,  which  is  found  in  muscle. 

97. — Prepare  myosin  from  lean  meat  by  chopping 
about  an  ounce  finely,  then  stirring  it  well  with  cold  water 
to  remove  the  albumin.  Filter  through  muslin,  and  repeat 
the  treatment  with  water  until  it  is  white  or  nearly  so. 
Squeeze  out  most  of  the  water  and  treat  the  residue  with 
a  10-per-cent.  solution  of  ammonium  chlorid.  For  thor- 
ough extraction  it  should  stand  several  hours,  but  enough 
for  testing  can  be  obtained  by  stirring  for  five  minutes. 
Filter  through  muslin,  then  through  paper.  The  filtrate 
contains  the  globulin  (myosin). 

98. — To  a  beaker  full  of  pure  water  add  a  little  of  the 
solution.  The  myosin  is  precipitated. 

99. — Heat  some  of  the  globulin  solution.  It  is  coagu- 
lated. 

100. — To  5  cubic  centimeters  of  the  myosin  solution  add  as 
much  saturated  ammonium  sulphate  solution.  A  precipitate  of 
the  globulin  forms.  Is  it  chemically  changed? 

101. — Saturate  10  cubic  centimeters  with  magnesium  sul- 
phate. The  globulin  is  precipitated,  but  dissolves  again  on  dilu- 
tion with  water.  Has  it  been  changed  chemically? 

102. — To  the  solution  of  myosin  add  enough  hydrochloric 
acid  to  make  it  contain  0.1  per  cent.  After  it  has  stood  a  few 
hours  syntonin,  an  acid  albumin,  has  been  formed.  If  the  change 
is  complete  it  does  not  coagulate  upon  heating.  If  there  is  a 
coagulum  it  is  some  of  the  unchanged  myosin.  In  this  case  filter 
it  out  and  test  the  filtrate  for  the  acid  albumin  by  carefully  neu- 
tralizing it  with  dilute  sodium  hydrate.  The  acid  albumin  which 
is  formed  from  the  globulin  by  the  dilute  acid  is  precipitated. 

103. — Try  the  general  tests  for  albuminous  substances  (xan- 
thoproteic,  biuret,  Millon's).  The  myosin  responds  to  all. 


46  THE  PKOTEINS. 

THE  ALBUMINATES. 

These  are  called  also  acid  and  alkali  albumins.  They 
are  formed  from  the  albuminous  substances  by  the  action 
of  acids  or  alkalies.  They  are  soluble  in  water  which  con- 
tains a  small  amount  of  acid  or  alkali,  but  are  not  in  neu- 
tral solution.  Consequently  they  are  precipitated  when 
their  solution  is  neutralized.  They  differ  from  the  glob- 
ulins by  being  insoluble  in  dilute  salt  solutions.  They  are 
not  coagulated  by  boiling.  The  alkali  albumin  has  the 
properties  of  an  acid,  giving  a  slight  acid  reaction.  Simi- 
larly the  acid  albumin  has  a  slight  alkaline  reaction.  They 
are  named  from  the  manner  in  which  they  are  produced, 
and  not  according  to  their  reaction. 


ACID   ALBUMIN. 

104. — Prepare  by  adding  dilute  HC1  to  a  solution  of 
egg-albumin  till  it  contains  0.1  per  cent,  of  the  acid. 
Allow  it  to  stand  an  hour,  at  about  body-temperature,  then 
filter  and  neutralize  with  very  dilute  sodium  hydrate,  being 
careful  not  to  add  an  excess,  as  this  would  dissolve  the 
precipitated  acid  albumin.  Wash  the  precipitate  in  water. 

105. — Notice  that  the  acid  albumin  is  soluble  in  acids, 
though  insoluble  in  water.  Make  a  solution  in  dilute  HC1 
and  boil.  It  is  not  coagulated. 

106. — Make  acid  albumin  by  the  action  of  strong 
HC1,  HN"03,  or  acetic  acid  on  serum-albumin  or  egg-albu- 
min, warming  if  necessary.  It  is  formed  very  quickly. 
Neutralize  a  portion  with  sodium  hydrate.  It  is  precipi- 
tated. Show  that  it  gives  the  xanthoproteic  reaction  and 
biuret  reaction,  though  it  is  not  precipitated  by  boiling. 


THE    ALBUMIN ATES.  47 


ALKALI   ALBUMIN. 

107. — Prepare  from  a  solution  of  albumin  by  warm- 
ing with  alkali,  like  sodium  hydrate. 

108. — The  solid  alkali  albumin  can  be  obtained  by  adding 
strong  sodium  hydrate,  drop  by  drop,  to  the  white  of  an  egg, 
stirring  continually.  No  more  must  be  added  after  it  has  become 
gelatinous,  as  it  will  then  dissolve.  Wash  the  solid  in  cold  water, 
in  which  it  is  insoluble,  though  it  is  soluble  without  difficulty  in 
warm  water.  (It  is  known  as  Lieberkiihn's  jelly.) 

109. — Observe  that  the  alkali  albumin  is  soluble  in 
dilute  acids  or  alkalies.  If  the  conversion  is  incomplete 
complete  solution  does  not  occur.  An  excess  of  acid  may 
precipitate  it. 

110. — Dissolve  some  of  the  solid  substance  in  hot 
water  or  use  the  solution  obtained  from  Experiment  107. 
Add  a  few  drops  of  phenolphthalein,  which  gives  a  red 
color,  showing  that  the  solution  is  alkaline.  Add  slowly 
dilute  acid  until  the  red  color  has  just  disappeared,  when 
the  solution  will  be  neutral.  The  alkali  albumin  is  pre- 
cipitated. If,  now,  more  acid  is  added,  the  precipitate  dis- 
solves. It  is,  consequently,  like  the  acid  albumin,  precipi- 
tated by  neutralizing. 

111. — Show  that  dissolved  alkali  albumin,  like  acid 
albumin,  is  not  coagulated  by  boiling. 

112. — Show  there  is  a  cleavage  of  egg-albumin  during 
the  formation  of  alkali  albumin  with  a  splitting  off  of  the 
loosely  combined  (cystin)  sulphur. 

First  heat  the  albumin  solution  with  sodium  hydrate 
for  several  minutes.  The  sulphur  which  has  been  split  off 
from  the  albumin  molecule  remains  in  the  solution  as  so- 
dium sulphid  and  does  not  discolor  a  piece  of  filter  paper 


48  THE   PROTEINS. 

which  is  moistened  with  lead  acetate  solution  and  held 
over  the  mouth  of  the  test-tube.  Upon  acidifying  the  hot 
solution  with  hydrochloric  acid  the  sulphur  previously 
liberated  is  given  off  as  hydrogen  sulphid  gas,  which  im- 
mediately turns  the  paper  brown. 

113. — In  the  same  manner  as  in  the  previous  experi- 
ment test  with  lead  paper,  the  gas  from  a  solution  of  albu- 
bin  when  boiled  with  hydrochloric  acid,  as  in  the  formation 
of  acid  albumin.  No  discoloration  appears;  that  is,  the 
albumin  has  not  been  decomposed  to  such  a  degree  as 
in  the  last  experiment. 

114. — The  thoroughly  washed,  solid  alkali  albumin  when 
pressed  upon  moist  litmus-paper  reddens  it,  and  its  solution  in 
calcium  hydrate  has  an  acid  reaction,  if  no  excess  of  the  latter  is 
present.  To  obtain  these  results,  however,  great  care  is  necessary 
to  free  it  from  the  alkali  used  in  its  preparation. 

PROTEOSES  AND  PEPTONES. 

The  peptones  are  the  final  products  of  the  digestive 
action  of  pepsin  upon  the  albuminous  compounds.  The 
albumoses  are  intermediate  products  between  the  albumi- 
nous compounds  and  the  peptones.  They  may  both  be 
formed  by  the  putrefaction  of  albuminous  substances. 

The  proteoses  give  the  general  reactions  of  the  al- 
buminous substances,  but  they  do  not,  like  the  former, 
coagulate  on  boiling.  They  are  distinguished  from  the 
peptones  by  giving  a  precipitate  with  nitric  acid  or  potas- 
sium ferrocyanid  acidified  with  acetic  acid.  They  are  also 
precipitated  by  saturating  their  solution  with  ammonium 
sulphate  or  sodium  chlorid,  then  acidifying.  They  diffuse 
with  difficulty  through  an  animal  membrane. 

The  peptones  do  not  give  any  of  these  reactions,  but 
respond  to  the  general  ones  of  albuminous  compounds,  espe- 


......  (r 


\j  t, 


THE   PROTEOSES   AND   PEPTONES.  49 

cially  the  biuret  test,  where  the  resulting  color  is  a  reddish 
pink.  They  do  not  coagulate  on  boiling,  and,  unlike  the 
other  albuminous  substances,  will  pass  through  a  parch- 
ment or  animal  membrane.  They  cannot  be  precipitated 
by  ammonium  sulphate  or  with  potassium  ferrocyanid. 
Tannin  or  alcohol  precipitates  them  from  their  solutions. 

A  number  of  different  classes  have  been  described,  two 
of  the  principal  ones  being  those  designated  by  the  prefixes 
— anti,  which  resist  the  action  of  ferments  and  are  not 
easily  decomposed  further;  and  Tiemi,  which  are  more 
easily  decomposed.  Thus  we  have  antialbumoses  and  hemi- 
albumoses,  and  antipeptones  and  hemipeptones.  The  other 
classes  differ  principally  in  their  solubilities. 

In  the  dry  state  the  proteoses  form  an  amorphous 
powder.  The  peptones  also  have  an  amorphous  form,  but 
are  extremely  hygroscopic,  dissolving,  to  a  resinous  mass, 
in  the  water  which  they  absorb  from  the  air.  Their  taste 
is  unpleasant. 

115.  PREPARATION  or  PROTEOSES. — Boil  for  twelve 
to  fifteen  hours  about  10  grammes  of  fibrin  or  coagulated 
egg-albumin  with  three  to  four  times  its  weight  of  4  per 
cent,  sulphuric  acid.  Keep  the  volume  constant  by  the  use 
of  an  inverted  condenser  or  by  the  addition  of  water.  The 
liquid  turns  violet,  then  brownish.  If  on  testing  a 
small  sample  by  neutralizing  with  sodium  hydrate  much 
acid  albumin  is  precipitated,  continue  the  heating  until 
such  a  precipitate  is  no  longer  produced  or  is  but  slight. 
Then  filter  off  any  insoluble  matter,  neutralize  with  am- 
monium hydrate,  and  saturate  the  liquid  with  ammonium 
sulphate.  The  proteoses  are  precipitated.  Filter  and 
wash  them  with  a  saturated  solution  of  the  last  reagent. 
Then  dissolve  the  proteoses  in  a  little  water,  place  this 
solution  in  a  dialyzer,  and  dialyze  until  the  outer  liquid 


50  THE   PROTEINS. 

gives  no  reaction  for  sulphates  with  barium  chlorid.  If 
the  water  is  warm  some  antiseptic  like  thymol  or  chloro- 
form must  be  used.  The  albumose  reactions  can  be  made 
with  this  solution  from  the  dialyzer. 

If  the  dry  proteoses  are  desired  concentrate  the  solu- 
tion to  a  small  bulk  and  precipitate  them  with  an  excess 
of  alcohol,  washing  with  the  same  after  filtration.  Dry 
in  a  vacuum  desiccator  over  sulphuric  acid. 

116. — Dissolve  some  of  the  proteose  in  water  and  show  that 
it  is  precipitated  by  potassium  ferrocyanid  in  a  solution  acidified 
with  acetic  acid,  avoiding  an  excess  of  the  former. 

117. — Show  that  a  solution  of  proteose  in  water  is  not  coagu- 
lated by  neutralizing  nor  boiling.  Nitric  acid  gives  a  white  pre- 
cipitate which  dissolves  on  heating  and  reappears  on  cooling.  (Dis- 
tinction from  albumin  and  albuminates.) 

118.  PREPARATION  OF  PEPTONE.— Digest  blood-fibrin  in  a 
neutral  solution  with  a  watery  extract  from  a  chopped  pancreatic 
gland  at  about  body-temperature.  If  it  is  allowed  to  stand  many 
hours,  add  a  few  crystals  of  thymol  to  prevent  putrefaction. 
Boil  after  solution  has  taken  place,  filter,  concentrate  by  boiling, 
and  saturate  while  boiling  with  ammonium  sulphate  to  precipitate 
the  albumoses.  Filter  these  out,  first  by  muslin,  then  by  filter- 
paper.  The  solution  may  be  used  for  testing  or  the  ammonium 
sulphate  may  be  removed  largely  by  evaporation  and  crystalliza- 
tion. The  remainder  can  be  removed  by  adding  first  barium 
hydrate,  then  barium  carbonate,  and  boiling,  until  a  portion  of 
the  filtrate  gives  no  precipitate  with  barium  chlorid. 

119. — Commercial  peptone  may  be  used  for  testing. 
Test  the  solution  of  peptones  by  adding  first  sodium  hy- 
drate, then  not  more  than  two  or  three  drops  of  copper  sul- 
phate (biuret  test).  A  red  or  pink  color  is  produced, 
which  is  characteristic  of  the  peptones.  If  too  much  of 
the  copper  solution  is  added,  the  color  is  bluish. 

120. — Place  some  of  the  solution  in  a  dialyzer  and 
leave  it  an  hour,  then  test  the  solution  outside  for  the 


INSOLUBLE   PROTEINS.  51 

presence  of  peptones.  They  will  be  found  to  have  passed 
through  the  membrane,  although  they  do  not  dialyze  rap- 
idly. 

121. — Show  that  tannic  acid  precipitates  peptones  in 
a  neutral  solution. 

122. — Show  that  the  peptones  are  not  precipitated  by 
potassium  ferrocyanid  acidified  with  acetic  acid,  as  are 
the  albumoses,  if  they  contain  none  of  the  latter. 

FIBRIN. 

Fibrin  is  formed  as  a  gelatinous  mass  when  fresh 
blood  coagulates.  If  the  blood  is  beaten  during  its  coag- 
ulation the  fibrin  collects  together  into  strings  as  elastic  as 
caoutchouc,  and  remains  so  as  long  as  it  is  moist.  It  can 
be  freed  from  the  blood  coloring  matter  by  washing  with 
water  or  a  salt  solution.  It  is  coagulated  by  heating  with 
water. 

COAGULATED  ALBUMIN. 

The  albumins  may  be  converted  into  the  coagulated 
form  by  heating  with  water  or  by  the  continued  action  of 
strong  alcohol.  This  is  insoluble  in  water,  but  can  be  dis- 
solved by  caustic  alkalies  or  by  heating  with  the  strong 
mineral  acids,  being  thereby  converted  into  alkali  or  acid 
albumins. 

COMPOUND  PROTEINS. 

This  class  of  substances  is  more  complex  than  the 
albuminous  substances.  They  can  all  be  decomposed  into 
albuminous  substances,  on  the  one  hand,  and,  on  the  other, 
bodies  which  are  not  albuminous.  Thus  the  albuminous 
compound  is,  in  hemoglobin,  united  with  the  hsematin 
molecule;  in  the  nucleins,  with  phosphoric  acid,  etc.  They 


52  THE   PROTEINS. 

can  be  considered,  then,  as  unions  of  an  albuminous  sub- 
stance with  some  other  substance.  Most  of  them  are  coagu- 
lated by  boiling. 

THE  MUCINS. 

Mucins  are  found  in  some  of  the  secretions  of  the  body, 
especially  in  those  of  the  mucous  membrane  and  saliva, 
and  also  as  a  constituent  of  the  tendons  and  umbilical  cord. 
In  their  composition  they  resemble  the  albuminous  sub- 
stances, but  contain  less  nitrogen.  Their  characteristic 
property  is  that  when  boiled  with  a  dilute  mineral  acid  they 
.are  decomposed,  giving  two  substances:  an  albuminous  com- 
pound and  a  compound  containing  little  or  no  nitrogen  and 
having  the  power  of  reduction,  as  is  shown  by  its  changing 
cupric  hydrate  to  cuprous  oxid  in  an  alkaline  solution. 
By  this  they  can  be  distinguished  from  all  similar  albu- 
minous compounds.  There  are  several  kinds  of  mucin, 
although  as  yet  they  are  not  well  differentiated  from  one 
another. 

The  mucins  are  colloid  substances,  insoluble  in  pure 
water,  but  soluble  in  small  amounts  of  dilute  alkalies,  such 
as  calcium  hydrate.  They  are  mucilaginous  and  can  be 
drawn  out  into  threads.  They  are  precipitated  by  the 
addition  of  acetic  acid,  if  neutral  salts  are  absent.  They 
are  not  coagulated  by  boiling,  but  give  many  of  the  reac- 
tions of  the  albumins.  Like  the  nucleoalbumins,  they  are 
acid  in  reaction. 

There  have  been  sometimes  included  with  the  mucins  a  class 
of  substances  which  resemble  them  in  being  decomposed  by  acids 
into  albuminous  substances  and  substances  with  the  power  of 
reduction,  but  which  are  not  precipitated  by  acetic  acid.  They 
are  more  properly  called  mucoids.  Such  are  the  pseudomucin, 
found  in  ovarial  liquids;  chondromucoid  of  cartilage,  and  a  few 
others. 


MUCIN.  NUCLEOALBUMINS.  53 

123.  PREPARATION  OF  MUCIN. — Mince  finely  a  sub- 
maxillary  gland  of  an  ox  and  extract  it  with  water.  Filter 
and  add  to  the  filtrate  strong  hydrochloric  acid  until  the 
liquid  contains  0.15  per  cent,  of  acid,  avoiding  an  excess. 
The  mucin  is  at  first  precipitated,  but  dissolves  again  upon 
stirring.  Then  add  two  or  three  volumes  of  water,  which 
will  precipitate  it.  Separate  it  from  the  liquid  by  filtration 
or  decantation  and  repeat  the  dissolving  and  precipitation 
as  before.  Wash  with  water  and,  if  the  dry  substance  is  de- 
sired, with  alcohol  and  ether. 

124. — Try  the  solubility  in  calcium  hydrate,  and  pre- 
cipitate from  this  solution  by  acetic  acid. 

125. — Boil  mucin  for  some  time  with  dilute  hydro- 
chloric acid,  and,  after  making  the  liquid  alkaline,  show 
by  Fehling's  test  that  there  is  a  reducing  body  present. 

126. — Show  that  solutions  of  mucin  in  an  alkali  will 
give  the  biuret  test. 

THE  NUCLEOALBUMINS. 

The  nucleoalbumins  occur  widely  distributed  in  the 
animal  and  vegetable  kingdoms,  forming  one  of  the  princi- 
pal constituents  of  protoplasm.  They  are  found  especially 
in  the  cell,  but  sometimes  in  the  secretions,  such  as  the  milk, 
which  contains  casein.  Some  authors,  however,  include 
in  the  class  of  nucleoalbumins  only  those  found  in  the  cell, 
and  exclude  such  as  the  casein  of  milk  and  the  vitellin  of 
eggs. 

Chemically  they  are  composed  of  the  same  elements 
as  albumin,  but  contain,  in  addition,  phosphorus  and  some- 
times iron.  The}''  resemble  in  their  properties  the  globu- 
lins and  alkali  albumins.  They  differ,  however,  in  contain- 
ing phosphorus.  They  are  also  insoluble  in  neutral  salt 


54  THE   NUCLEOALBUMINS. 

solutions,  unlike  the  globulins,  and  differ  from  the  alkali 
albumins  by  being  decomposed  by  the  action  of  gastric  juice 
into  an  albumin,  which  is  digested,  and  an  indigestible 
phosphorus  compound  called  nuclein.  The  nucleoalbumins 
may  be  considered,  then,  as  compounds  of  a  nuclein  and  an 
albuminous  substance. 

The  nucleoalbumins  are  insoluble  in  water  or  dilute 
acids.  They  can  be  dissolved  in  alkaline  solutions.  They 
have  the  properties  of  dibasic  acids,  as  is  shown  by  the  fact 
that  the  solution  in  an  alkali,  where  not  too  much  of  the 
alkali  has  been  used,  has  a  slight  acid  reaction.  The  acid 
reaction  is  also  shown  by  their  setting  free  the  carbonic 
acid  of  carbonates.  The  nucleoalbumins  can  be  precipi- 
tated from  their  alkaline  solutions  by  acidifying.  The 
acid  removes  the  alkali  with  which  they  are  united  and  sets 
the  nucleoalbumin  free  as  an  insoluble  substance.  This 
may  be  shown  in  the  precipitation  of  the  casein  in  milk. 

The  nucleoalbumins  are  soluble  in  strong  acetic  acid 
or  an  excess  of  hydrochloric  acid,  being  at  the  same  time 
decomposed,  nuclein  being  set  free  and  the  albumin  being 
converted  into  acid  albumin.  They  are  also  decomposed 
and  coagulated  by  suspending  the  free  nucleoalbumin  in 
water  and  boiling. 

In  general,  being  compounds  of  the  albuminous  sub- 
stances, the  nucleoalbumins  respond  to  the  same  tests. 

127.  PKEPAKATION  OF  CASEIN. — Dilute  about  100 
cubic  centimeters  of  milk  with  400  cubic  centimeters  of 
water,  and  precipitate  the  casein  until  the  liquid  above 
is  nearly  clear  by  adding  acetic  acid  drop  by  drop,  avoiding 
an  excess.  Filter  and  wash  with  water.  If  fat-free  sub- 
stance is  desired,  it  must  be  extracted  in  an  extraction- 
apparatus  with  ether.  For  many  tests  this  is  not  neces- 
sary. The  casein  can  be  to  some  degree  purified  by  dis- 


CASEIN.  55 

solving  in  dilute  ammonia  and  reprecipitating  with 
acid. 

128. — Test  the  casein  for  nitrogen  and  sulphur  in  the 
same  manner  as  albumin  was  tested  (Experiments  72  and 
73). 

129. — Test  it  also  for  phosphorus  by  mixing  about 
a  gramme  of  the  dry  substance  with  equal  parts  of  sodium 
carbonate  and  potassium  nitrate  and  fusing  in  a  porcelain 
crucible.  After  cooling,  dissolve  the  mass  in  water,  acidify 
strongly  with  nitric  acid,  and  add  ammonium  molybdate. 
A  yellow  precipitate,  at  once  or  after  warming,  shows  the 
presence  of  phosphoric  acid. 

130. — Try  the  solubility  of  casein.  It  is  insoluble  in 
water,  but  soluble  in  alkalies.  Its  alkaline  solution  is  not 
coagulated  by  heat.  With  lime  water  it  forms  a  milky 
solution.  Its  solutions  give  the  biuret  reaction  and  Millon's 
reaction. 

131. — Demonstrate  the  acid  nature  of  freshly  precipi- 
tated casein  by  dissolving  it  in  a  very  dilute  solution  of 
sodium  carbonate.  If  not  quite  enough  of  the  latter  is  used 
to  produce  a  complete  solution  and  if  the  mixture  is  then 
filtered,  the  nitrate  will  have  an  acid  reaction  showing 
that  the  sodium  casein  which  it  contains  has  some  of  the 
properties  of  an  acid  salt.  Boiling  causes  no  coagulation, 
but  the  casein  can  be  set  free  as  a  precipitate  if  hydro- 
chloric acid  is  added  to  strong  acid  reaction.  (Compare 
with  results  of  acidifying  solutions  of  soap  or  of  soluble 
carbonates.) 

132. — If  the  rennin  ferment  is  at  hand  or  can  be  pre- 
pared, test  with  it  a  solution  of  casein  in  lime-water.  The 
casein  is  changed  into  paracasein  calcium  (cheese)  which 
is  only  slightly  soluble  in  water. 


56  THE   NUCLEINS. 

133. — Make  a  gastric  juice  by  adding  a  few  grains  of  pepsin 
to  0.2-per-cent.  HCI1  and  digest  in  it  for  some  time  at  the  tempera- 
ture of  the  body  some  casein.  The  nucleoalbumin  is  decomposed 
into  two  substances:  an  albumin,  which  is  dissolved,  and  a 
nuclein,  a  compound  rich  in  phosphorus,  which  remains.  Filter  out 
the  nuclein,  and  test  the  nitrate  with  the  biuret  test  for  the 
albuminous  compound. 


THE  NUCLEINS. 

The  nucleins  occur  partly  combined  with  albuminous 
substances  as  nucleoalbumins,  partly  free  in  the  nucleus  of 
the  cell.  They  are  composed  of  phosphoric  acid  united 
with  an  albumin,  or  sometimes  in  addition  with  a  nuclein 
base,  such  as  adenin,  guanin,  xanthin,  or  hypoxanthin. 
Besides  the  elements  found  in  vhe  albumin  they  contain 
phosphorus  and  sometimes  iron.  The  iron  in  the  haamo- 
globin  is  probably  obtained  by  the  animal  organism  from 
such  compounds  as  these.  We  have  no  absolute  proof  that 
the  common  salts  of  iron  can  be  assimilated  by  the  animal 
organism.  One  of  the  nucleins  which  may  furnish  iron  to 
the  animal  body  is  haamatogen,  found  in  the  yelk  of  eggs. 
The  nucleins  may  be  divided  into  two  classes: — 
1.  Those  which,  when  acted  on  by  hot  acids  or  alka- 
lies, take  up  water  and  give,  as  decomposition-products, 
phosphoric  acid  and  an  albumin.  They  have  been  called 
paranucleins  or  pseudo-nudeins.  Such  a  one  is  contained 
in  casein.  It  can  be  set  free  by  digesting  with  gastric  juice 
from  the  albumin  with  which  it  is  united.  Haamatogen  is 
also  of  this  class,  and  probably  furnishes  the  iron  for  the 
haemoglobin  of  the  young  bird. 


1  This  strength  of  acid  can  be  obtained  by  diluting  concentrated 
hydrochloric  acid  with  150  times  its  volume  of  water. 


THE   NUCLEINS.  57 

2.  The  second  class  is  decomposed  by  dilute  acids  or 
alkalies,  giving,  besides  phosphoric  acid  and  an  albumin, 
one  or  more  of  the  nuclein  bases,  also  called  xanthin  bases, 
or  purin  bases,  xanthin,  guanin,  etc.  These  nucleins  are, 
then,  combinations  of  albuminous  substances  with  nu- 
cleinic  acids.  This  class  is  found  principally  in  the  nucleus 
of  the  cell  and  has  been  called  nuclear,  or  nucleous  nu- 
cleins. 

The  nucleins  are  not  attacked  by  gastric  juice,  and 
this  is  used  to  separate  them  from  the  albuminous  com- 
pounds, which  can  be  digested  by  it.  They  are  insoluble 
in  water  and  dilute  acids,  but  soluble  in  alkalies.  They 
give  the  biuret  and  Millon's  tests  in  consequence  of  the 
albumin  which  they  contain. 

134. — Mix  50  grammes  of  compressed  or  brewers'  yeast  with 
200  grammes  of  water,  and  allow  the  yeast  to  subside.  Pour 
off  the  water  and  to  the  residue  add  O.o-per-cent.  potassium 
hydrate  solution;  stir,  and,  after  waiting  a  few  minutes  to  dissolve 
the  nucleins,  filter  and  acidify  with  hydrochloric  acid.  Filter  out 
the  precipitated  nucleins,  wash  with  HC1,  then  hot  alcohol.  Dry 
over  sulphuric  acid.  Try  solubility  in  acids  and  alkalies,  also  biuret 
and  Millon's  tests.  The  residue  gained  by  fusion  with  sodium  car- 
bonate and  potassium  nitrate  gives  a  yellow  precipitate  with  am- 
monium molybdate,  showing  the  presence  of  phosphoric  acid. 

A  few  of  the  nucleins,  both  animal  and  vegetable,  contain 
iron,  and  these  are  the  principal  source  of  the  iron  in  the  animal 
body,  our  food-materials  containing  no  inorganic  compounds  of 
iron.  Hsematogen  is  an. example  of  such  nucleins.  It  is  found  in 
the  yelk  of  hen's  eggs  united  with  an  albuminous  substance, 
which  can  be  split  off  by  digestion. 

The  iron  in  such  organic  combinations  as  nuclein  does  not 
respond  to  the  ordinary  chemical  tests  until  the  compound  has 
been  decomposed  by  chemical  agents  or  other  means.  Reagents 
like  ammonium  sulphid  and  potassium  ferrocyanid  decompose 
it  very  slowly,  whereas  they  act  immediately  upon  the  inorganic 
compounds  of  iron  and  not  at  all  upon  hsematin.  The  nucleins 


58  H^lMATOGEN.  THE  ALBUMINOIDS. 

which  contain  iron  are  important  from  their  being  probably  the 
origin  of  the  animal  iron  compounds. 

H^IMATOGEN. 

135. — Prepare  from  the  yelk  of  an  egg.  Shake  the  yelk  in 
a  wide-mouth,  glass-stoppered  bottle  with  two  or  three  times  its 
volume  of  alcohol;  allow  it  to  stand  and  when  it  has  settled  pour 
off  the  alcohol.  Repeat  this  operation  twice,  then  extract  in  the 
same  manner,  or,  better,  in  an  extraction  apparatus  with  ether 
until  the  residue  is  white.  Digest  this  in  artificial  gastric  juice 
(made  as  in  Experiment  133).  The  nuclein,  haematogen,  remains. 

136. — Dissolve  a  portion  in  ammonia.  Test  for  iron  by 
ammonium  sulphid.  At  first  there  is  no  color,  but  after  a  time 
the  solution  turns  greenish  and,  in  twenty-four  hours,  black,  as 
the  iron  is  gradually  set  free  from  the  compound.  In  the  same 
manner  test  with  potassium  ferrocyanid.  The  Prussian  blue  is 
formed  slowly,  differing  thus  from  its  production  with  the  ordi- 
nary iron  salts. 

137. — Add  to  the  nuclein  some  hydrochloric  acid,  then,  after 
neutralizing  with  ammonia,  test  for  iron  as  before.  The  acid  has 
decomposed  the  nuclein,  so  that  the  tests  are  obtained  imme- 
diately. 

THE  ALBUMINOIDS. 

The  albuminoids  are  found  in  the  insoluble  form, 
mostly  in  the  bones  of  the  body  or  the  parts  which  are 
used  for  protection.  They  resemble  the  albuminous  sub- 
stances, giving  many  of  the  same  products  when  they  are 
decomposed,  differing,  however,  in  other  respects.  They 
are  not  easily  attacked  by  the  reagents  which  dissolve  and 
decompose  albuminous  compounds.  Only  the  collagen  and 
its  derivative,  gelatin,  with  possibly  elastin,  are  digestible. 

COLLAGEN. 

This  substance  is  found  in  the  animal  body  in  the 
connective  and  cartilaginous  tissues,  tendons,  and  bones. 


GELATIN.  59 

That  from  the  bones  was  formerly  called  ossein.  It  is  com- 
posed of  the  same  chemical  elements  as  the  albumins,  but 
contains  a  little  more  oxygen.  It  is  probably  an  oxidation- 
product  of  some  of  the  albumins.  It  is  insoluble  in  water, 
but  by  boiling  with  water  it  is  converted  into  gelatin  or 
glue.  By  the  action  of  tannic  acid  collagen  is  changed  to 
a  form  which  does  not  putrefy.  This  is  the  action  which 
takes  place  when  leather  is  tanned. 

GELATIN. 

Gelatin  may  be  considered  as  the  hydrate  of  collagen, 
as  it  is  formed  by  the  union  of  collagen  with  water.  It 
can  be  changed  back  into  collagen  by  heating  for  some 
time  at  130°.  It  swells  up  in  cold  water  and  dissolves 
when  the  water  is  warmed.  On  allowing  the  solution  to 
cool  it  gelatinizes,  or  becomes  a  semi-solid.  It  acts  in  this 
respect  oppositely  to  albumin,  which  is  soluble  in  the  cold, 
but  becomes  a  solid  by  the  action  of  heat. 

After  it  has  been  boiled  a  long  time  with  water  it  is 
decomposed,  and  does  not  gelatinize  on  cooling,  peptone 
being  formed.  The  sulphur  of  gelatin  is  united  in  the 
molecule  in  a  different  manner  from  that  of  the  albumin 
molecule,  as  is  proved  by  its  decomposition-products. 

Gelatin  is  decomposed  by  the  gastric  juice,  giving 
products  similar  to  those  from  albumin.  It  has  been 
found,  however,  that  it  cannot  take  the  place  of  the  al- 
buminous materials  of  food,  though  it  is  of  value  when 
used  with  them. 

Gelatin  does  not  give  all  the  reactions  of  the  albumi- 
nous substances,  although  it  does  give  the  same  results  with 
some  of  them.  Like  the  albuminous  compounds,  it  gives 
a  purple  color  with  the  biuret  test;  it  is  precipitated  by 


60  GELATIN. 

picric  acid,  by  mercuric  chlorid  in  the  presence  of  sodium 
chlorid  and  hydrochloric  acid,  by  tannic  acid  in  the  pres- 
ence of  sodium  chlorid,  and  by  saturation  with  ammonium 
sulphate.  On  the  other  hand,  it  is  not  coagulated  by  boil- 
ing; it  is  not  precipitated  by  mineral  acids;  and  it  does  not 
give  a  brown  color  when  warmed  with  an  alkaline  solution 
of  lead,  as  albumin  does,  the  sulphur  being  apparently  too 
firmly  united  to  be  split  off  and  form  lead  sulphid.  It 
does  not  give  the  xanthoproteic  reaction  when  pure. 

138. — Prepare  collagen  from  bone  by  dissolving  out 
the  mineral  constituents  with  dilute  hydrochloric  acid 
(HC1,  1  part;  water,  8  parts)  until  they  are  flexible,  then 
wash  out  the  acid.  Notice  that  the  collagen  is  not  soluble 
in  dilute  acid  nor  cold  water.  To  remove  all  the  albumins 
it  may  be  necessary  to  soak  awhile  in  5-per-cent.  NaOH 
solution,  then  wash  again. 

139. — Convert  the  collagen  into  gelatin  by  boiling  it 
a  few  minutes  with  water.  Notice  that  it  gelatinizes  upon 
cooling  the  solution,  especially  after  standing. 

140. — Boil  a  portion  of  the  solution  for  some  time, 
and  notice  that  it  is  thus  decomposed,  so  that  it  will  not 
form  a  jelly  upon  cooling. 

141. — Test  a  portion  of  the  gelatin  solution  with  the 
biuret  test.  It  gives  a  purple  color  like  albumin. 

142. — Show  that  it  is  precipitated  by  tannic  acid  in 
the  presence  of  NaCl. 

143. — Show  that  gelatin  is  not  precipitated  by  nitric  or  other 
mineral  acids,  but  is  by  saturation  with  ammonium  sulphate  and 
also  by  mercuric  chlorid  in  the  presence  of  HC1  and  NaCl. 

144. — Show  that  gelatin  contains  sulphur  by  heating  with 
dry  sodium  carbonate  in  the  reducing  flame,  then  testing  with 
sodium  nitroprussid  as  in  the  case  of  albumin  (Experiment  74), 
but  that  it  gives  no  black  sulphid  of  lead  when  heated  in  a  solution 
of  lead  acetate  in  an  excess  of  sodium  hydrate  (Experiment  73). 


ELASTIN.  KERATIN.  61 


ELASTIC. 

Elastin  occurs  in  the  connective  tissues, — in  the  cervical  liga- 
ment (ligamentum  nuchae)  very  abundantly.  It  differs  from  most 
of  the  proteins  in  containing  no  sulphur,  except  possibly  some 
that  is  loosely  combined  with  the  molecule.  In  the  moist  state  it 
is  very  elastic;  when  dry  it  is  hard  and  brittle.  By  the  action 
of  the  digestive  ferments  it  is  decomposed  into  bodies  called  elas- 
toses,  similar  to  the  albumoses.  It  gives  the  general  reactions  of 
the  proteins. 

145. — Prepare  elastin  from  the  cervical  ligament  of  an  ox  by 
cutting  it  into  thin  slices  and  boiling  it  for  several  days  to  remove 
the  gelatin.  Boil  then  with  1-per-cent.  potassium  hydrate  for 
several  hours,  afterward  with  water.  Repeat  the  boiling  with  10- 
per-cent.  acetic  acid;  then  let  it  stand  twenty-four  hours  in  5- 
per-cent.  hydrochloric  acid.  Wash  with  water,  boil  with  95-per- 
cent, alcohol,  and  extract  with  ether  to  remove  the  fat.  For  the 
complete  removal  of  the  latter  more  than  a  week  may  be  required. 

146. — Try  the  tests  used  for  sulphur  in  albuminous  sub- 
stances, and  see  that  it  is  not  present. 


KERATIN. 

The  keratins  are  the  chief  constituent  of  the  horny  part  of 
the  epidermis,  of  hair,  horns,  nails,  feathers,  etc.  They  contain 
a  large  amount  of  sulphur, — 4  or  5  per  cent., — a  part  of  which 
is  so  loosely  united  that  it  is  set  free  by  boiling  water.  It  is 
owing  to  this  sulphur  that  the  salts  of  lead,  silver,  and  some  other 
metals  act  as  hair-dyes,  the  sulphur  uniting  with  the  metal  to 
form  a  dark-colored  sulphid.  The  keratin  is  not  at  all  attacked 
by  the  gastric  or  pancreatic  juices.  It  is  decomposed  when  heated, 
giving  the  odor  of  burnt  horn.  It  is  insoluble  in  water,  and  gives 
the  xanthoproteic  and  Millon's  reactions. 

147. — Prepare  keratin  by  boiling  some  horn-shavings  with 
water  and  then  digesting  them  in  succession  in  a  dilute  solution  of 
pepsin  containing  0.2-per-cent.  HC1,  and  a  trypsin  solution.  Wash 
with  water,  alcohol,  and  ether. 


62  FERMENTATION. 

148. — Boil  keratin  with  sodium  hydrate,  filter,  and  test  the 
filtrate  for  sulphur  by  lead  acetate.  It  produces  a  black  precipi- 
tate of  lead  sulphid. 

149. — Show  that  keratin  responds  to  the  xanthoproteic  and 
Millon's  tests. 

FEBMENTATIOK 

By  fermentation  we  mean  the  decomposition  of  an 
organic  substance  into  simpler  and  more  stable  molecules, 
the  agent  which  causes  the  change  being  itself  unaffected. 
The  agents  are  living  organisms  or  are  formed  by  such 
organisms.  The  living  ferments — such  as  the  yeast-plant 
or  bacteria — are  often  called  the  organized  ferments. 
They  have  the  power  of  reproduction  and  are  composed  of 
cells.  The  non-living  ferments  are  known  as  the  unor- 
ganized ferments,  or  enzymes.  They  may  be  excreted  by 
the  organized  ferments  or  secreted  by  living  cells,  which 
latter  is  the  case  with  the  digestive  ferments.  They  are 
not  reproductive  and  act  outside  of  the  cell  where  they 
were  formed. 

The  enzymes  of  the  animal  cell  exist  in  the  cells  in  an 
inactive  condition,  called  zymogens,  but  become  active 
after  standing  exposed  to  the  atmosphere  or  being  brought 
in  contact  with  certain  chemical  compounds.  The  enzymes 
contain  nitrogen  and  from  their  properties  are  apparently 
identical  with  the  proteins.  They  are  indiffusible  and 
soluble  in  glycerin  and  in  water.  They  can  be  mechanic- 
ally removed,  without  decomposition,  from  their  solutions 
by  forming  precipitates  therein,  to  which  they  adhere,  also 
by  saturating  the  solutions  with  ammonium  sulphate.  A 
low  temperature  stops  their  action  and  they  are  all  killed 
below  100°  if  moisture  is  present.  Most  enzymes  act  best 
at  about  38°  C.  The  ferment  is  not  destroyed,  but  its 


ORGANIZED   FERMENTA.  63 

action  is  stopped,  by  a  large  accumulation  of  its  own 
products. 

The  organized  ferments  contain  albumin,  fat,  cellu- 
lose, and  some  inorganic  salts.  They  survive  a  high  tem- 
perature better  than  the  enzymes,  but  are  killed  at  100° 
except  certain  spore  forms.  Moisture  is  necessary  for  them 
to  act. 

As  is  the  case  with  the  enzymes,  a  sufficient  amount 
of  their  products  stops  their  further  action.  This  is  the 
effect  of  alcohol  upon  the  yeast-plant. 

150. — Add  a  little  yeast  to  a  dilute  solution  of  cane- 
sugar  in  water  and  keep  it  for  some  time  at  the  body-tem- 
perature. 

Test  the  solution  with  Trommels  test.  The  copper 
compound  is  reduced  by  the  glucose  and  Ia3vulose  which 
have  been  formed  from  the  sucrose  by  the  invertin  of  the 
yeast.  If  allowed  to  stand  a  long  time  the  glucose  is 
changed  by  the  yeast  to  alcohol  and  carbon  dioxid. 

151. — Stir  a  little  compressed  yeast  into  lukewarm 
water  in  a  test-tube,  and  after  it  has  stood  a  few  minutes, 
add  a  few  drops  of  chloroform  and  mix  thoroughly  by 
shaking.  Fill  the  rest  of  the  tube  now  with  dilute  cane- 
sugar  solution  and  let  it  stand  inverted  for  twenty-four 
hours  in  a  warm  place,  as  in  Experiment  31.  The  alco- 
holic enzyme  (zymase)  does  not  act  in  presence  of  chloro- 
form, so  that  there  is  no  alcoholic  fermentation  with  a 
formation  of  carbon  dioxid. 

152. — Test  the  liquid  with  Trommer's  test.  It  re- 
sponds to  the  test,  showing  that  the  inverting  enzyme  is 
iwt  destroyed,  but  has  decomposed  the  cane-sugar,  as  be- 
fore, to  glucose  and  lavulose. 


64  FERMENTATION. 

153. — Separate  the  mucous  membrane  from  the  muscular 
coating  of  a  pig's  stomach;  chop  finely  and  allow  to  stand  several 
hours  with  two  or  three  times  its  weight  of  dilute  phosphoric  acid 
(1  per  cent.).  Filter  and  to  the  filtrate,  which  contains  the  pepsin, 
add  lime-water  until  the  reaction  is  alkaline.  The  calcium  phos- 
phate which  falls  carries  down  the  pepsin  with  it.  Filter  and  dis- 
solve the  precipitate  in  dilute  HC1.  Place  in  a  dialyzer,  changing 
the  water  outside  frequently.  The  acids  and  salts  diffuse  out, 
leaving  the  pepsin  inside  the  dialyzer,  as  can  be  proved  by  adding 
it  to  0.2-per-cent.  HC1  and  seeing  that  it  will  digest  fibrin. 

154. — Test  a  part  of  the  precipitated  pepsin  which  has  been 
obtained  with  the  calcium  phosphate  for  nitrogen.  This  may  be 
done,  after  washing  with  water,  by  drying  the  precipitate,  still 
mixed  with  calcium  phosphate,  then  mixing  with  twice  as  much 
soda-lime  and  testing  in  a  dry  tube.  The  nitrogen  is  converted 
into  ammonia,  which  may  be  recognized  by  the  odor  and  by  its 
action  on  red  litmus-paper. 

155. — To  a  part  of  the  dialyzed-pepsin  solution  obtained  in 
Experiment  153  add  finely  powdered  ammonium  sulphate,  stirring 
meanwhile  as  long  as  it  dissolves.  The  pepsin  is  precipitated  like 
the  albuminous  compounds.  Filter,  dissolve  the  precipitate  in  a 
0.2-per-cent.  HC1  solution,  and  show  that  it  will  digest  fibrin. 

156. — Collect  some  saliva  in  a  test-tube,  place  the 
latter  in  a  beaker  of  water,  and  raise  the  temperature  of 
the  water  to  65°  or  70°  C.  Keep  it  at  this  point  for  five 
minutes.  Then  let  a  little  of  it  stand  a  few  minutes  with 
a  starch  solution,  testing  afterward  with  Trommer's  test. 
N~o  glucose  is  produced,  the  ferment,  ptyalin,  having  been 
destroyed  by  the  heat. 

157. — Collect  a  considerable  quantity  of  saliva,  and 
put  it  into  two  tubes.  Quickly  cool  one  nearly  to  freezing 
and  warm  the  other  to  body-temperature.  Add  to  these 
an  equal  amount  of  starch  solution.  Allow  the  action  to 
proceed  for  five  minutes,  then  raise  them  both  to  the  boil- 
ing-point to  stop  fermentation.  Determine  the  amount  of 


ORGANIZED  FERMENTA.  65 

sugar  formed  by  Fehling's  quantitative  test  (Experiment 
33),  or,  approximately,  by  the  subnitrate  of  bismuth  test 
(Experiment  30).  There  has  been  little  or  no  fermenta- 
tion in  the  cold  liquid. 

158. — In  the  same  manner  cool  another  portion  of 
the  saliva  and,  after  ten  minutes,  warm  to  body-tempera- 
ture and  show  by  its  decomposing  starch  that  the  ferment 
is  not  destroyed  by  the  cold. 

159.  PREPARATION  OF  LACTIC  ACID  BY  FERMENTATION. — 
In  150  cubic  centimeters  of  boiling  water  dissolve  30  grammes  of 
cane-sugar  and  add  about  30  milligrammes  of  tartaric  acid.     Let 
the  solution  stand  two  days,  then  add  40  cubic  centimeters  of  sour 
milk  and  about  half  a  gramme  of  old  cheese.     After  the  addition 
of  15  grammes  of  zinc  oxid  allow  the  mixture  to  stand  ten  days  at 
a  temperature  of  40°  to  50°,  with  repeated  stirrings.     At  the  end 
of  that  time  heat  to  boiling,  filter  while  hot,  and  allow  to  cool. 
Zinc  lactate  will  crystallize  out  on  cooling  if  the  solution  is  suffi- 
ciently concentrated.     If  it  is  not  it  should  be  allowed  to  evapo- 
rate to  a  smaller  volume.     It  may  be  purified  by  recrystallizing. 
The  acid  can  be  obtained  by  dissolving  the  lactate  in  water  and 
decomposing  by  hydrogen  sulphid  gas.    Filter  off  the  zinc  sulphid, 
evaporate  the  filtrate  to  a  syrup,  and  after  it  is  cold  extract  the 
acid  by  dissolving  it  in  ether.1     When  the  ether  has  evaporated 
the  acid  will  remain.    It  may  be  preserved  for  testing  in  the  gastric 
juice  tests. 

160.  PREPARATION  OF  BUTYRIC  ACID. — Make  a  mixture  as 
for  the  preparation  of  lactic  acid,  or  use  a  part  of  that  mixture. 
Allow  the  fermentation  to  go  on  as  before,  but  for  three  or  four 
weeks.      In  that   time   bubbles   of  hydrogen   and   carbon  dioxid 
appear  and,  after  removing  the  zinc,  butyric  acid  is  found  in  the 
ether.    It  can  be  identified  by  its  acid  reaction  and  characteristic 
odor. 


1  Remember  that  the  vapors  of  ether  are  extremely  inflam- 
mable. 


66  THE   SALIVA. 

161.  PREPARATION  OF  KEPHYB. — The  preparation  of  the 
ferment  requires  several  days.  First  stir  the  kephyr-grains  in  a 
little  lukewarm  water  and  decant  the  latter.  Then  cover  them 
with  ten  times  their  weight  of  milk  which  has  been  boiled  and 
cooled.  Let  them  stand  for  twenty-four  hours  at  about  20°  C., 
then  pour  off  the  milk,  rinse  the  grains  with  a  little  water,  and 
repeat  the  digestion  with  milk  for  twenty-four  hours  as  before. 
This  should  be  continued  until  the  milk  has  an  acid  reaction  and 
the  grains  rise  to  the  upper  part  of  the  liquid,  which  may  require 
five  or  six  days.  After  this  the  grains  may  be  allowed  to  stand 
with  a  moderate  quantity  of  milk,  which  becomes  filled  with  the 
organized  ferment.  At  the  end  of  twenty- four  hours  this  is  filtered 
through  muslin,  and  the  filtrate  added  to  ten  or  fifteen  times  its 
volume  of  previously  boiled  and  cooled  milk.  The  whole  is  poured 
into  a  clean  strong  bottle,  the  cork  is  tied  in,  and  it  is  allowed  to 
stand  two  or  three  days  at  a  temperature  below  15°  C.,  when  the 
fermentation  is  complete.  The  grains  on  the  muslin  can  be  used 
an  indefinite  number  of  times  in  the  same  manner. 

162.— Test  the  acid  reaction  of  the  kephyr  by  litmus-paper 
(lactic  acid) ;  also  show  the  presence  of  alcohol  by  the  iodoform 
test  (Experiment  32). 


THE  SALIVA. 

The  saliva  is  a  mixture  of  the  secretions  of  the  paro- 
tid, submaxillary,  and  sublingual  glands  with  that  of  the 
glands  of  the  membrane  of  the  month.  Its  reaction  is 
normally  faintly  alkaline.  The  mixed  saliva  is  a  colorless, 
more  or  less  viscid  liquid,  often  opalescent.  On  standing 
it  deposits  calcium  carbonate  as  a  film  on  the  surface.  Ex- 
amined microscopically  it  is  seen  to  contain  epithelial  cells 
from  the  membrane  of  the  mouth,  air-bubbles  held  by 
viscid  liquid,  and  salivary  corpuscles  which  resemble  the 
lymph-corpuscles.  Bacteria  are  abundant. 


THE   SALIVA. 

The  normal  mixed  saliva  contains 


I.  Inorganic 


II.  Organic 


Carbonates   ]          [  magnesium. 
Chlorids         !          !   calcium. 
Sulphates.     [          |   potassium. 
Nitrites  [  sodium. 

Sulphocyanate  of  potassium. 
Albumin. 
Mucin. 
Ptyalin. 


Nitrites  and  sulphocyanates  (sulphocyanids)  are  often 
absent.  The  latter  are  most  frequently  found  in  the  saliva 
of  smokers.  The  ptyalin  has  the  power  to  convert  boiled 
starch  into  dextrin,  maltose,  and  glucose.  It  is  not  able 
to  penetrate  the  granule  of  the  unboiled  starch,  or  does  so 
very  slowly,  differing  in  this  respect  from  the  correspond- 
ing ferment  of  the  pancreas.  Its  presence  can  be  detected 
by  mixing  the  saliva  with  about  ten  times  its  volume  of 
a  solution  of  boiled  starc,h,  keeping  it  awhile  at  body-tem- 
perature, and  after  a  few  minutes  testing  for  sugar.  The 
ptyalin  acts  best  at  about  40°,  and  is,  therefore,  not  the 
same  ferment  as  the  diastase  of  malt,  which  decomposes 
starch  most  rapidly  at  a  temperature  of  55°.  Ptyalin  is 
destroyed  by  acids,  even  as  dilute  as  the  0.2-per-cent.  hy- 
drochloric acid  of  the  gastric  juice.  It  is,  however,  prob- 
able that  it  acts  for  some  time  in  the  stomach  before  the 
acid  penetrates  the  mass  of  food  in  a  large  enough  quan- 
tity to  stop  the  fermentation. 

The  secretion  is  influenced  by  the  nervous  system.  It 
can  be  increased  by  mechanical  means,  like  chewing  a 
pebble  or  a  piece  of  rounded  glass  in  the  mouth;  by 
chemical  action,  such  as  touching  the  tongue  with  a  crys- 


68  THE   SALIVA. 

tal  of  tartaric  acid  or  filling  it  with  the  vapor  of  ether  or 
acetic  acid ;  or  by  electrical  excitation.  In  collecting  saliva 
for  testing  it  should  be  accomplished  without  trying  to 
hasten  its  flow  by  suction  with  the  tongue  as  this  increases 
the  amount  of  secretion  from  the  mucous  membrane  and 
so  dilutes  the  secretion  of  the  salivary  glands. 

The  composition  of  the  saliva  is  changed  by  certain 
pathological  conditions.  The  amount  is  diminished  in  all 
febrile  conditions^  also  in  diabetes  and  often  in  nephritis. 
It  is  increased  by  the  action  of  some  medicinal  substances, 
like  the  mercury  compounds,  pilocarpine,  and  others ;  also 
by  anything  which  causes  irritation  or  inflammation  of  the 
glands.  Urea  has  been  found  in  it  abundantly  during 
nephritis.  The  reaction  becomes  acid  in  fevers  and  in 
diabetes,  and  this  sometimes  happens  also  after  long-con- 
tinued talking. 

163. — Collect  for  examination  some  saliva  by  letting 
it  flow  into  the  mouth  without  swallowing.  Excite  the 
flow  by  chewing  a  piece  of  soft  paraffin.  Notice  the  indi- 
cation of  mucin  in  the  viscidity,  as  well  as  the  lasting  foam 
after  beating  it  with  a  glass  rod. 

164. — Let  a  portion  stand  exposed  to  the  air,  and 
notice  the  separation  of  calcium  carbonate  as  a  white  film 
or  turbidity. 

165. — Test  a  portion  for  potassium  sulphocyanate  by 
adding  to  it  a  very  dilute  solution  of  ferric  chlorid.  A 
red  color  indicates  the  sulphocyanate.  This  is  immediately 
decolorized  by  the  addition  of  a  few  drops  of  mercuric 
chlorid  solution. 

166. — Test  for  nitrites  with  a  few  drops  of  a  starch 
solution  acidified  with  a  little  dilute  sulphuric  acid  and 
containing  a  small  amount  of  potassium  iodid.  A  nitrite 
immediately  gives  a  blue  color. 


SALIVARY   TESTS.  69 

167. — Test  for  albuminous  substances  by  the  xan- 
thoproteic  reaction. 

168. — Test  for  mucin  by  adding  to  the  clear  saliva 
acetic  acid  drop  by  drop.  The  mucin  separates  in  white, 
stringy  flakes.  They  may  be  washed  with  water  and  tested 
by  the  mucin  reactions. 

169.  PREPARATION  OF  PTYALIN. — Collect  a  large  amount  of 
saliva  and  acidify  with  phosphoric  acid.  Then  add  milk  of  lime 
until  the  liquid  has  a  faint  alkaline  reaction.  The  phosphoric  acid 
is  precipitated  as  calcium  phosphate  and  carries  the  ptyalin  down 
with  it.  Filter  and  allow  the  water  to  drain  off  without  washing. 
Place  the  precipitate  in  a  beaker  and  add  not  more  water  than 
the  original  amount  of  the  saliva.  Stir  it  well  and  filter.  This 
removes  the  ptyalin  from  the  calcium  phosphate,  and  it  goes  into 
the  nitrate.  Add  to  the  nitrate  an  excess  of  alcohol.  A  white 
precipitate  will  separate,  which  is  ptyalin  mixed  with  inorganic 
salts.  To  free  it  from  these,  dissolve  in  a  little  water  and  pre- 
cipitate with  absolute  alcohol.  Repeat  this  operation  if  necessary. 
Dry  it  over  sulphuric  acid. 

170. — Test  the  aqueous  solution  of  ptyalin.  It  is  not  precipi- 
tated by  nitric  acid  like  albumin,  nor  does  it  give  the  xantho- 
proteic  reaction.  It  can  be  precipitated  after  a  time  by  basic  lead 
acetate,  the  filtrate  being  without  action  on  starch. 

171. — Try  the  reaction  of  saliva  to  litmus  and  to 
phenol-phthalein.  Normally  it  is  alkaline  with  the  former 
and  neutral  with  the  latter.  Since  alkaline  salts  affect 
litmus  while  they  do  not  change  the  color  of  phenol- 
phthalein,,  it  is  evident  that  here  no  free  alkalies  are  pres- 
ent, but  that  the  reaction  is  due  to  alkaline  salts. 

172. — In  a  test-tube  treat  about  5  cubic  centimeters 
of  a  1  per  cent,  solution  of  starch  (made  as  in  6)  with  a 
few  drops  of  saliva  and  set  it  in  water  of  body  temperature. 
From  time  to  time  remove  a  drop  and  test  it  with  iodin 
solution.  Notice  the  change  of  color  from  the  blue  first  ob- 


70  THE  SALIVA. 

tained  through  purple  and  red  to  yellow.  Then  the  liquid 
will  reduce  Trammer's  reagent.  Hence  the  starch  has 
undergone  hydrolysis,  passing  through  the  dextrins  to 
sugar  (maltose). 

173. — Try  the  same  experiment  with  unboiled  starch. 
Glucose  is  not  formed,  or,  if  at  all,  only  after  a  long  time 
and  in  small  amounts. 

174.  —  Try  the  effect  of  dilute  acids  by  diluting  1 
cubic  centimeter  of  concentrated  hydrochloric  acid  with 
150  cubic  centimeters  of  water,  then  adding  to  the  saliva 
an  equal  volume  of  the  dilute  acid.  This  makes  the  acidity 
of  the  whole  about  the  same  as  that  of  the  gastric  juice. 
Let  the  acidified  saliva  act  on  boiled  starch  as  before.  No 
glucose  is  produced. 

175. — Place  2  cubic  centimeters  of  saliva  in  each  of  seven  test- 
tubes,  adding  2  cubic  centimeters  of  starch  solution;  both  of  these 
are  to  be  measured  by  a  pipette.  From  a  burette  containing  0.1- 
per-cent.  HC1  run  into  the  separate  tubes  4  cubic  centimeters,  2 
cubic  centimeters,  1  cubic  centimeter,  0.5  cubic  centimeter,  0.2  cubic 
centimeter,  and  0.1  cubic  centimeter.  The  last  tube  receives  no 
acid.  Let  them  all  stand  in  water  at  body  temperature.  Observe 
the  changes  in  appearance  of  the  solutions.  At  intervals  remove 
a  drop  of  liquid  from  each  tube  by  the  pipette  and  test  it  with  a 
drop  of  iodin,  making  notes  of  the  speed  of  digestion.  When 
a  blue  color  is  no  longer  produced,  Trommer's  test  for  maltose 
can  be  made.  Calculate  the  strength  of  acid  contained  in  each 
tube. 

176. — Place  about  4  cubic  centimeters  of  saliva  in  each  of 
four  test-tubes.  Add  from  a  burette  or  cylindrical  pipette  con- 
taining 1-per-cent.  sodium  carbonate  1  cubic  centimeter,  0.5  cubic 
centimeter,  0.25  cubic  centimeter,  and  0.1  cubic  centimeter  re- 
spectively, warming  with  boiled  starch  in  a  beaker  of  water  and 
noting  results  as  before.  The  alkali  retards  digestion,  but  does 
not  prevent  it. 

177. — Try  the  effect  of  free  and  combined  acid  on  ptyalin  as 
follows:  In  each  of  two  tubes  mix  about  4  cubic  centimeters  of 


THE   GASTRIC   JUICE.  71 

saliva  with  a  few  drops  of  a  strong  solution  of  albumin.  Use  for 
an  indicator  two  or  three  drops  of  tropseolin,  which  turns  red 
with  free  acid,  but  remains  yellow  if  the  acid  is  combined  with 
a  proteid.  To  each  tube  add  very  dilute  hydrochloric  acid,  to  the 
first  enough  to  produce  a  permanent  red  color,  to  the  second  not 
enough  for  this.  Try  their  digestive  power  on  starch  as  before. 
The  trace  of  free  acid  has  destroyed  the  enzyme;  the  acid  com- 
bined with  the  albumin  hinders  digestion  but  does  not  wholly 
prevent  it. 

178. — In  each  of  a  series  of  test-tubes  place  2  to  3  cubic  centi- 
meters of  saliva,  add  half  as  much  of  the  starch  solution  and  a 
few  drops  of  the  following  antiseptics:  Chloroform,  sodium 
fiuorid,  salicylic  acid,  alcohol,  mercuric  chlorid,  copper  sulphate. 
Let  the  tubes  stand  in  water  at  38 °,  occasionally  removing  a  drop 
and  testing  this  for  unaltered  starch  with  iodin  solution.  Since 
mercury  salts  form  a  compound  with  iodin  it  will  be  necessary  to 
use  more  of  the  reagent  in  this  instance.  When  no  more  remains 
Trommels  test  will  show  the  presence  of  maltose.  The  antisep- 
tics do  not  prevent  digestion  except  the  metallic  salts  which  unite 
with  the  albuminous  substances. 


THE  GASTRIC  JUICE. 

The  gastric  juice,  secreted  by  the  glands  of  the  stom- 
ach, differs  from  the  other  digestive  fluids  in  having  an 
acid  reaction.  It  is  a  clear,  thin  liquid  containing,  as  in- 
organic constituents,  principally  the  chlorids  and  phos- 
phates of  the  alkalies  and  of  calcium  and  magnesium. 
There  is  more  hydrochloric  acid  than  can  unite  with  the 
bases,  and  this  must  consequently  he  in  the  free  state.  The 
most  important  of  the  organic  substances  are  two  enzymes, 
or  unorganized  ferments:  pepsin  and  rennin. 

The  acidity  of  the  gastric  juice  is  caused  principally 
by  the  free  hydrochloric  acid,  but  may  be  at  times  due 
to  the  acid  phosphates,  or  to  the  organic  acids:  lactic, 
butyric,  and  acetic.  The  hydrochloric  acid  and  acid  phos- 


72  THE   GASTRIC   JUICE. 

phates  are  present  in  the  normal  juice.  The  lactic  acid 
may  be  found  in  the  first  stages  of  digestion,  especially 
when  the  food  contains  much  of  the  carbohydrates,  but 
is  not  normally  found  after  digestion  has  proceeded  more 
than  half  an  hour.  The  acetic  and  butyric  acids  are  not 
normally  present. 

The  free  hydrochloric  acid  appears  to  be  formed  from 
the  chlorids  which  are  taken  with  the  food.  Its  forma- 
tion has  been  explained  as  due  to  the  action  of  disodium 
phosphate  upon  calcium  chlorid  and  also  to  the  decomposi- 
tion of  sodium  chlorid  by  a  weak  acid,  like  carbonic  acid. 
Neither  explanation,  however,  is  perfectly  satisfactory.  A 
free  acid  is  necessary  for  the  digestion  of  the  nitrogenous 
foods  by  the  pepsin,  and  this  is  one  of  the  offices  of  the 
hydrochloric  acid.  Recent  researches  have  indicated  that 
one  of  its  most  important  functions  is  the  prevention  of 
fermentation  in  the  stomach.  The  mineral  acids  have 
antiseptic  powers  even  in  such  dilution  as  that  of  the  hy- 
drochloric acid  in  the  gastric  juice;  that  is,  from  0.2  to 
0.3  per  cent.  Such  a  solution  will,  for  several  days,  pre- 
vent putrefaction  in  animal  matter,  like  chopped  meat, 
which  would  otherwise  soon  commence  to  decay.  It  will 
also  destroy  the  bacteria  of  many  infectious  diseases, 
though  some  of  these  are  not  affected  when  in  the  form 
of  spores. 

The  effects  of  increased  fermentation  are  seen  in  cer- 
tain pathological  conditions  where  the  secretion  of  hydro- 
chloric acid  is  diminished  or  stopped.  They  are  especially 
noticeable  in  the  case  of  food  containing  large  quantities 
of  carbohydrates.  The  sugar  which  is  formed  by  the  saliva 
may  be  changed  by  the  ferments  into  different  acids: — 

C6H1206  —  2C3H603. 

cuoel  sS  lactic  acid 


GASTRIC   DIGESTION.  73 

By  further  fermentation  the  lactic  acid  undergoes  this 
change: — 

2C3H603  =  C3H7C02H  +  2C02  +  2H2. 

butyric  acid 

The  glucose  may  be  fermented  to  alcohol: — 
C6H1206  =  2C2H5OH  +  2C02 

alcohol 

and  this  be  converted  to  acetic  acid: — 

C2H5OH  +  02  =  CH3C02H  +  H20. 

acetic  acid 

The  so-called  "heart-burn,"  which  is  one  of  the  accompanying 
symptoms  of  gastric  fermentation  is  caused  by  the  eructations  of 
gas  carrying  the  acids  up  into  the  throat,  where  they  cause  irrita- 
tion of  the  mucous  membrane.  The  methods  of  treatment  based 
upon  administration  of  hydrochloric  acid,  creasote,  or  other  anti- 
septic substances  is  not  for  the  purpose  of  removing  the  acids,  but 
to  stop  the  fermentation.  Treatment  with  alkaline  substances  like 
magnesia  or  sodium  bicarbonate  neutralizes  the  acids,  but  does  not 
prevent  fermentation. 

The  acid  phosphates  may  be  present  in  the  normal 
gastric  juice.  When  present  they  increase  the  acidity  of  the 
juice  or  cause  an  acid  reaction  in  the  absence  of  free  acids. 
An  example  is  the  potassium  compound,  KH2P04,  which 
is  found  in  the  stomach  after  meat  has  been  eaten. 

The  pepsin  can  be  obtained  from  the  gastric  juice  or 
mucous  membrane  of  the  stomach  in  a  number  of  ways. 
Like  most  of  the  animal  ferments,  it  can  be  extracted  from 
the  membrane  by  glycerin,  and  this  glycerin  solution  can 
be  preserved  for  any  length  of  time.  If  it  is  to  be  used 
immediately,  water  containing  about  0.2-per-cent.  hydro- 
chloric acid  can  be  employed  for  extraction.  If  the  dry 
substance  is  desired,  it  may  be  thrown  down  with  finely- 


74  THE   GASTRIC   JUICE. 

divided  precipitates  of  other  substances,  as  is  the  case  with 
many  of  the  ferments. 

The  pepsin  thus  obtained  is  probably  not  the  pure 
substance.  It  is  a  white  or  yellowish-white,  amorphous 
powder  or  scale  when  dried.  It  is  hygroscopic  in  the  air 
and  has  a  slightly  saline  or  acidulous  taste,  with  no  offen- 
sive odor.  It  is  soluble  in  about  100  parts  of  water,  but 
more  easily  on  the  addition  of  hydrochloric  acid.  Because 
of  its  reactions  pepsin  is  generally  regarded  as  a  protein. 
Its  molecular  composition  is  unknown. 

Pepsin  is  inactive  in  neutral  or  alkaline  liquids,  but 
in  slightly  acid  fluids  it  dissolves  coagulated  albuminous 
compounds,  with  the  formation  of  proteoses  and  peptones. 
It  acts  most  rapidly  with  hydrochloric  acid,  though  others 
may  be  used  instead.  The  best  strength  of  acid  for  the 
purpose  varies  with  the  kind  of  material  to  be  digested  from 
0.1  per  cent,  to  0.3  per  cent,  of  hydrochloric  acid.  Pepsin 
from  warm-blooded  animals  digests  best  at  38°.  It  is 
destroyed  by  heating  the  solution.  Its  action  is  hindered 
by  the  presence  of  the  products  of  digestion,  but  if  these 
are  removed  as  fast  as  they  are  formed  it  will  change  to 
the  soluble  form  many  thousand  times  its  weight  of  albu- 
minous material. 

The  second  ferment  of  the  gastric  juice,  rennin,  is 
always  present  in  human  gastric  juice  under  normal  condi- 
tions. It  exists  in  the  mucous  membrane  in  the  form  of  a 
zymogen,  which  is  sometimes  inactive  until  it  is  set  free 
by  an  acid.  Hence  if  it  is  extracted  by  water  it  may  not 
give  its  characteristic  reaction.  This  is  especially  true  in 
the  case  of  birds. or  fish.  This  characteristic  reaction  is  the 
coagulation  of  milk  or  casein  in  a  neutral  or  faintly-alka- 
line solution  in  which  calcium  salts  are  present.  It  does 
not  give  the  albumin  reactions  when  in  a  pure  state.  Ben- 


GASTRIC   DIGESTION.  75 

nin  is  more  easily  destroyed  by  heating  its  solution  than  is 
pepsin.  The  methods  of  obtaining  it  are  similar  to  those 
employed  with  pepsin. 

The  gastric  juice  acts  only  upon  the  nitrogenous  con- 
stituents of  the  food.  The  albuminous  substances  are  first 
changed  into  acid  albumin  by  the  free  acid;  then  this  is 
decomposed  by  the  pepsin  and  hydrochloric  acid,  forming 
proteose,  which  passes  into  peptone.  Since  the  process  is 
a  continuous  one,  all  these  products  may  be  found  in  the 
stomach  at  the  same  time,  though  in  the  first  stages  the 
acid  albumin  is  in  excess  and  at  the  last  the  peptones. 
The  connective  tissues  are  digested  by  the  gastric  juice, 
though  the  proteolytic  ferment  of  the  pancreas  does  not 
dissolve  them.  The  membranes  which  surround  the  fat- 
cells  are  also  dissolved,  setting  the  fat  free.  Thus  the  food 
is  changed  into  chyme:  a  pulpy  mass  which  can  be  readily 
penetrated  by  the  intestinal  fluids.  In  the  first  stages  of 
digestion  the  saliva  may  continue  to  act  on  the  starchy 
materials,  and  during  the  first  fifteen  to  twenty  minutes 
there  is  a  formation  of  lactic  acid,  which  disappears  after 
this  time.  Milk  is  coagulated,  partly  by  the  acid,  partly 
by  the  rennin.  The  absorption  of  the  peptones  commences 
in  the  stomach,  but  the  digestion  is  not  completed  here,  the 
chyme  passing  through  the  pylorus  into  the  intestine. 

179.  PREPARATION  OF  PEPSIN. — Separate  the  mu- 
cous membrane  of  a  pig's  stomach  from  the  muscular  tis- 
sue. After  rinsing  it  with  water  chop  it  finely.  Make  a 
dilute  solution  of  hydrochloric  acid  by  the  addition  of  1 
cubic  centimeter  of  concentrated  acid1  to  150  cubic  centi- 
meters of  water.  This  will  contain  about  0.2  per  cent, 
of  the  pure  acid.  Extract  the  pepsin  from  the  chopped 


1  The  U.  S.  P.  acid  containing  about  32-per-cent.  HC1. 


76  THE    GASTRIC   JUICE. 

membrane  by  means  of  this  dilute  acid.  To  obtain  a  strong 
solution  of  pepsin  it  is  best  to  let  it  stand  for  twenty-four 
hours  in  a  cool  place,  though  it  will  be  found  in.  the  solu- 
tion in  a  very  short  time.  The  rennin,  which  is  dissolved 
at  the  same  time,  is  destroyed  by  the  pepsin  on  standing. 
Filter  the  liquid  through  muslin,  then,  if  necessary, 
through  paper.  This  solution  can  be  used  directly  for 
digestive  experiments  or  for  preparing  the  purified  pepsin. 


180. — Purified  pepsin  can  be  obtained  by  precipitating  it  with 
some  substance  which  is  thrown  down  as  a  finely-divided  precipi- 
tate. To  effect  this,  neutralize  the  hydrochloric  acid  solution  of 
pepsin  in  the  preceding  experiment  and  acidify  with  phosphoric 
acid,  or  extract  the  pepsin  from  the  membrane  with  water  acidified 
slightly  with  phosphoric  acid.  Precipitate  the  phosphoric  acid 
solution  by  adding  lime-water  or  milk  of  lime  until  the  liquid  is 
alkaline.  Filter  off  the  precipitate,  which  contains  the  pepsin  mixed 
with  calcium  phosphate.  Dissolve  in  water  with  the  addition  of 
hydrochloric  acid.  Remove  the  salts  by  dialysis.  The  pepsin  does 
not  pass  through  the  membrane.  A  purer  form  is  obtained  by 
repeating  the  solution  in  acid  and  precipitation  with  lime-water 
or  by  precipitating  with  alcohol  before  dialyzing.  If  the  dry  sub- 
stance is  desired,  the  drying  must  be  at  a  low  temperature.  This 
may  be  done  over  sulphuric  acid  in  a  vacuum. 

181. — PROOF  OF  THE  EXISTENCE  OF  ZYMOGENS. — This  is 
based  upon  the  facts  that  dilute  acids  convert  the  zymogen  into 
the  enzyme  and  that  dilute  alkalies  destroy  the  latter,  but  not  the 
former. 

Dilute  5  cubic  centimeters  of  the  glycerin  extract  of  the  gas- 
tric mucosa  from  a  recently  killed  animal  in  a  test-tube  (A)  with 
an  equal  volume  of  0.2-per-cent.  hydrochloric  acid.  In  another 
tube  (B)  dilute  the  glycerin  extract  with  as  much  water.  Warm 
both  for  15  minutes  at  38°;  then  add  to  each  one-half  of  its  volume 
of  1-per-cent  sodium  carbonate  and  warm  at  38°  for  half  an  hour. 
Carefully  neutralize  them  and  add  enough  hydrochloric  acid  to 
make  0.2  per  cent.  Test  for  the  presence  of  pepsin  by  a  shred  of 
fibrin.  It  is  found  in  B,  but  not  in  A. 


GASTRIC   DIGESTION.  77 

Where  the  membrane  has  been  for  some  time  exposed  to  the 
air  before  extracting  the  active  enzyme  may  have  been  formed 
and  consequently  no  difference  be  perceptible. 

182. — To  make  a  pepsin  solution  which  may  be  pre- 
served, though  it  is  somewhat  impure,  chop  the  mucous 
membrane  finely,  as  in  the  previous  methods,  and  after 
squeezing  it  in  a  piece  of  cloth  to  remove  the  water  as 
far  as  possible,  cover  it  with  two  or  three  times  its  volume 
of  glycerin  and  let  it  stand  for  a  week.  Filter  it  through 
a  piece  of  muslin,  pressing  out  the  glycerin.  This  gives  a 
permanent  solution  which  can  be  used  at  any  time  for 
digestive  experiments. 

183. — In  each  of  a  series  of  test  tubes  standing  in  a  beaker  of 
water  at  38°  place  a  little  pepsin  solution  and  a  shred  of  fibrin. 
Then  add  to  (a)  lactic  acid  up  to  0.8  per  cent.;  (b)  oxalic  acid  up 
to  1  per  cent.;  (c)  hydrochloric  acid  to  10  per  cent;  (d)  hydro- 
chloric acid  to  0.2  per  cent. ;  but  wrap  the  fibrin  tightly  with  fine 
thread  so  that  it  cannot  swell,  (e)  0.2-per-cent.  hydrochloric  acid. 
Note  the  speed  of  digestion. 

184. — Make  an  artificial  gastric  juice  by  preparing 
a  0.2-per-cent.  solution  of  hydrochloric  acid  as  in  Experi- 
ment 179,  and  adding  to  this  a  small  quantity  of  the 
glycerin  solution  of  pepsin.  The  solution  made  directly 
with  the  dilute  acid  can  be  used  if  it  is  fresh.  To  this  add 
a  small  handful  of  washed  fibrin.  If  this  is  not  at  hand, 
boiled  egg-albumin  may  be  substituted  for  it,  though  in 
this  case  the  digestion  is  slower.  Warm  the  mixture  care- 
fully to  about  body-temperature  and  keep  it  at  this  point 
until  the  albuminous  substance  has  dissolved.  Notice 
that  the  edges  first  become  transparent,  then  are  dissolved. 
Too  high  heat  will  destroy  the  ferment.  The  fibrin  should 
be  nearly  digested  in  half  an  hour.  It  may.be  left  for 


78  THE    GASTRIC   JUICE. 

several  days  without  danger  of  putrefaction  provided  it 
contains  0.2  per  cent,  of  the  free  acid. 

When  the  fibrin  has  nearly  or  quite  disappeared  boil 
one-half  the  liquid  to  coagulate  any  unchanged  albumin- 
ous compound,  and  filter.  The  filtrate  contains  the  prod- 
ucts of  digestion:  acid  albumin,  albumoses,  and  peptones. 
The  proportion  of  each  varies  with  the  time  of  digestion. 
The  amount  of  peptones  is  usually  small  until  the  pepsin 
has  acted  for  a  considerable  time. 

185. — Precipitate  the  acid  albumin  by  neutralizing 
carefully  with  very  dilute  sodium  hydrate.  Filter  it  out 
and  test  as  in  Experiments  104  and  105. 

186. — From  the  filtrate  precipitate  the  albumoses  by 
saturating  the  solution  with  ammonium  sulphate  by  the 
aid  of  heat.  Filter  and  test  the  precipitated  albumoses  as 
in  Experiments  116  and  117. 

187. — The  filtrate  from  the  albumoses  contains  the 
peptones.  Test  it  according  to  the  tests  used  in  Experi- 
ments 119,  121,  and  122.  When  the  biuret  test  is  made 
in  presence  of  ammonium  salts  a  large  excess  of  sodium 
hydrate  must  be  used.  Allow  the  second  half  of  the  diges- 
tion to  proceed  for  a  week  before  testing  for  peptones,  add- 
ing hydrochloric  acid  if  it  is  exhausted. 

188.  PREPARATION  OF  RENNIN. — Extract  the  chopped  mu- 
cous membrane  with  0.2-per-cent.  hydrochloric  acid,  as  in  Experi- 
ment 179.  Both  rennin  and  pepsin  go  into  solution.  Stir  the 
membrane  well  in  the  acidified  water,  but  do  not  let  it  stand  a 
very  long  time,  as  the  rennin  is  digested  in  an  acid  solution  by  the 
pepsin.  Neutralize  carefully,  add  a  small  amount 'of  magnesium 
carbonate,  and  shake  well.  The  pepsin  adheres  to  the  carbonate 
and  can  be  filtered  out  with  it.  Shake  the  filtrate  with  another 
portion  of  magnesium  carbonate,  filter,  and  repeat  the  operation 
until  the  pepsin  is  removed,  which  can  be  shown  by  the  failure  of 
an  acidified  portion  of  the  filtrate  to  dissolve  fibrin.  The  filtrate 
containing  the  rennin  should  coagulate  milk  in  a  few  minutes. 


RENNIN.  79 

189. — In  order  to  purify  rennin,  precipitate  it  with  basic 
lead  acetate  and  filter.  Wash  the  precipitate,  then  suspend  it 
in  water,  and  acidify  slightly  with  sulphuric  acid.  Filter,  and  to 
the  filtrate,  which  contains  the  rennin,  add  a  solution  of  stearin 
soap.  The  latter  is  decomposed  by  the  acid,  stearic  acid  being  set 
free  as  an  insoluble  precipitate.  This  carries  the  rennin  with  it. 
Filter  and  place  the  precipitate,  with  a  small  amount  of  water,  in 
a  glass-stoppered  funnel.  Add  ether,  shake,  and  after  the  ether 
has  separated  above  the  watery  solution  draw  off  the  latter.  The 
fatty  acids  from  the  decomposed  soap  have  remained  in  the  ether, 
leaving  the  rennin  dissolved  in  the  water. 

190. — Test  the  solution  or  a  specimen  of  normal  or 
artificial  gastric  juice  for  rennin  by  exactly  neutralizing, 
then  adding  it  to  an  equal  volume  of  milk  in  a  test-tube. 
Place  the  tube  in  a  beaker  of  water  at  body-temperature. 
If  rennin  is  present  the  casein  will  be  coagulated  in  twenty 
or  thirty  minutes. 

A  solution  of  casein  may  be  used  instead  of  milk,  but 
in  order  to  make  it  coagulate  a  small  amount  of  a  calcium 
salt  must  be  added. 

For  demonstrating  the  properties  of  rennin  the  com- 
mercial product,  which  is  sometimes  sold  under  the  name 
of  junket,  may  be  employed. 

191. — To  5  cubic  centimeters  of  milk  add  1  cubic 
centimeter  of  rennin  solution  and  keep  at  body-tempera- 
ture. Note  the  time  of  coagulating. 

192.- — Eepeat  the  above,  adding  2  cubic  centimeters 
of  ammonium  oxalate  solution  to  the  mixture  of  milk  and 
rennin.  This  precipitates  calcium  from  its  solutions. 
How  does  it  influence  coagulation  ?  Will  it  now  coagulate 
if  calcium  chlorid  is  added  ? 

193. — Is  the  rennin,  after  it  has  been  boiled,  capable 
of  coagulating  milk?  What  does  this  show  as  to  its 
nature  ? 


80  THE   GASTRIC   JUICE. 

194.  VALUATION  OF  PEPSIN  (U.  S.  P.,  1900).— Mix  9  cubic 
centimeters  of  10  per  cent,  hydrochloric  acid  with  291  cubic  centi- 
meters of  distilled  water,  and  in  150  cubic  centimeters  of  this  liquid 
dissolve  0.1  gramme  of  pepsin.  Immerse  and  keep  a  hen's  egg, 
which  should  be  fresh,  in  boiling  water  during  15  minutes.  Then 
remove  it  and  place  in  cold  water.  Separate  the  coagulated 
albumin  from  the  pellicle  and  yelk  and  rub  it  through  a  clean 
sieve  having  40  meshes  to  the  linear  inch.  Reject  the  first  portion 
passing  through  the  sieve.  Weigh  10  grammes  of  the  second, 
cleaner  portion,  place  it  in  a  flask  of  a  capacity  of  about  100  cubic 
centimeters,  then  add  20  cubic  centimeters  of  the  diluted  acid  and 
with  a  glass  rod  disintegrate  the  albumin.  Rinse  the  rod  with  15 
cubic  centimeters  of  the  dilute  acid,  add  5  cubic  centimeters  of  the 
pepsin  solution,  cork  and  shake.  Place  the  flask  in  a  water -bath 
kept  at  a  temperature  of  52 °C.  for  two  and  one-half  hours,  shaking 
every  ten  minutes,  then  remove  and  add  50  cubic  centimeters  of 
cold,  distilled  water.  Transfer  to  a  narrow  glass  cylinder  and 
allow  to  stand  half  an  hour.  The  deposit  of  undissolved  albumin 
should  not  be  more  than  1  cubic  centimeter.  Trustworthy  results, 
particularly  in  comparative  trials  will  be  obtained  only  if  the 
temperature  be  strictly  maintained  and  if  the  contents  of  the 
flasks  be  agitated  uniformly  and  in  equal  intervals  of  time. 

The  relative  proteolytic  power  of  pepsin  stronger  or  weaker 
than  that  described  above  may  be  determined  by  ascertaining, 
through  repeated  trials,  how  much  of  the  above  pepsin  solution 
will  be  required  exactly  to  dissolve  10  grammes  of  coagulated  and 
disintegrated  albumin  under  the  conditions  given  above.  Divide 
15,000  by  this  quantity  expressed  in  cubic  centimeters  to  ascertain 
how  many  parts  of  egg  albumin  one  part  of  pepsin  will  digest. 

In  recent  years  the  composition  of  the  gastric  juice  and 
its  variations  in  disease  are  being  more  and  more  thoroughly 
studied  and  the  results  of  the  observations  made  use  of  in 
clinical  work.  The  only  obstacle  to  the  general  adoption 
of  these  tests  is  probably  the  difficulty  of  obtaining  the 
fluid  from  the  stomach. 

With  a  little  experience,  the  collection  of  the  gastric  juice 
for  testing  can  be  easily  accomplished.  In  order  to  excite  the  flow 


GASTRIC   TESTS.  81 

a  test-meal  should  be  given.  This  should  be  rather  simple,  and 
may  consist  of  bread  or  rolls  with  weak  tea  without  milk.  A 
large  amount  of  food  rich  in  albuminous  materials  should  be 
avoided,  as  the  peptones  resulting  from  its  digestion  interfere  with 
some  of  the  tests.  With  such  a  meal  digestion  is  at  its  height  in 
about  an  hour,  and  the  collection  should  be  made  one  to  one  and 
a  half  hours  after  eating. 

The  apparatus  for  withdrawing  the  juice  is  an  elastic  rubber 
tube  about  a  yard  in  length,  having  a  number  of  small  perforations 
in  the  end  or,  if  a  large  opening,  it  should  be  on  the  side,  to  avoid 
injury  to  the  mucous  membrane  by  suction.  The  perforated  end 
is  passed  slowly  down  the  oesophagus  until  it  reaches  the  fundus 
of  the  stomach,  known  by  the  resistance  to  its  further  passage. 
The  flow  of  the  juice  through  the  siphon  is  best  started  by  press- 
ure upon  the  stomach,  the  outer  end  of  the  tube  being  held  lower 
than  the  stomach  and  over  a  collecting  vessel.  By  this  means  the 
juice  is  not  diluted. 

Sometimes  it  is  recommended  to  start  the  siphon  by  filling 
it  with  water  by  means  of  a  funnel,  then  when  it  is  full,  pinching 
the  top  of  the  tube  to  close  it  and  quickly  lowering  it.  The  first 
part  of  the  liquid  which  runs  out  is  water  and  should  be  thrown 
away,  collecting  only  the  last  part.  Otherwise  the  amounts  of 
constituents  found  must  be  corrected  for  the  water,  which  dilutes 
the  juice.  The  addition  of  water,  however,  prevents  accurate  quan- 
titative tests. 

Before  testing  the  juice  it  should  be  filtered  through  a 
plaited  filter,  keeping  it  covered  to  avoid  loss  of  water  or 
acid  by  evaporation. 

The  tests  which  are  usually  made  upon  the  gastric 
juice  are  for: — 

1.  Eeaction. 

2.  Acid  phosphates. 

3.  Hydrochloric  acid. 

(Lactic. 
Butyric. 
Acetic. 
5.  Pepsin. 


82  THE   GASTRIC    JUICE. 

Others  which  are  not  so  important,  but  the  presence 
of  which  may  sometimes  be  significant,  are  for: — 

1.  Starch. 

2.  Albuminous  compounds. 

3.  Eennin. 

4.  Blood-coloring  matters. 

Quantative  tests  are  valuable  in  the  cases  of: — 

1.  Total  acidity. 

2.  Hydrochloric  acid,  free   and  combined. 

3.  Organic  acids. 

The  reaction  of  normal  gastric  juice  is,  of  course,  acid. 
In  some  pathological  conditions  it  becomes  neutral  or  alka- 
line. Litmus-paper  can  be  used  for  the  test. 

To  determine  the  total  acidity  of  the  juice  filter  it, 
keeping  it  covered  to  prevent,  as  much  as  possible,  evapora- 
tion; then  measure  accurately  10  cubic  centimeters  with  a 
pipette  and  place  it  in  a  beaker.  Add  to  this  a  few  drops 
of  an  alcoholic  solution  of  phenol-phthalein,  which  serves 
as  an  indicator  to  tell  whether  the  liquid  is  acid  or  alkaline 
during  the  determination,  being  red  with  alkalies  and  color- 
less with  acids.  Then  add  slowly  from  a  burette  a  solution 
containing  4  grammes  of  sodium  hydrate  to  the  liter,  stir- 
ring continually,  until  the  liquid  is  a  faint  pink  color  which 
remains  on  standing  a  few  minutes.  Enough  of  the  stand- 
ard alkali  has  then  been  added  to  neutralize  the  acid  sub- 
stances present.  Eead  off  this  amount  from  the  burette. 
If  no  gastric  juice  is  at  hand,  a  solution  for  experimental 
purposes  can  be  made  of  a  mixture  of  the  above  acids  after 
greatly  diluting  them. 

The  acid  phosphates  may  be  normally  present,  but 
cannot  perform  the  functions  of  the  hydrochloric  acid  in 


GASTRIC   TESTS.  83 

digestion.  Hence  it  is  important  to  be  able  to  detect  them. 
They  can  be  distinguished  from  free  acids  from  the  fact 
that  they  are  not  neutralized  by  calcium  carbonate  in  the 
cold  as  the  free  acids  are.  When,  therefore,  a  gastric  juice 
is  neutralized  by  adding  to  it  finely  powdered  calcium  car- 
bonate, like  precipitated  chalk  (which  must  itself  be  neu- 
tral), no  acid  phosphates  are  present,  but  the  reaction  was 
due  to  free  acids.  If  the  color  of  the  litmus-paper  is  ob- 
scured by  the  excess  of  calcium  carbonate,  this  may  be  rinsed 
off  with  distilled  water.  If  the  reaction  remains  acid  after 
calcium  carbonate  has  been  added,  acid  phosphates  are  pres- 
ent. Their  amount  can  be  determined  by  finding  the  total 
acidity  of  10  cubic  centimeters,  then  adding  calcium  car- 
bonate to  15  cubic  centimeters,  filtering  and  determining 
the  acidity  of  10  cubic  centimeters  of  the  filtrate.  This 
latter  is  due  to  the  phosphates.  The  difference  between 
the  two  is  the  amount  of  the  free  acids. 

195. — Test  the  reaction  of  the  acid  phosphates1  to 
litmus-paper. 

196. — Show  that  they  are  not  neutralized  by  calcium 
carbonate  in  the  cold. 

197.— Show  that  the  dilute  free  acids  (both  HC1  and 
lactic)  can  be  so  neutralized. 

For  a  short  time  after  food  has  been  taken  hydro- 
chloric acid  may  be  wanting,  or  present  only  in  traces  in 
the  gastric  juice,  without  any  pathological  significance, 
but  in  one  to  three  hours  after  a  meal  it  should  be  found 
in  larger  amounts.  It  is  then,  under  normal  conditions, 
about  0.1  to  0.3  per  cent,  of  the  weight  of  the  juice. 

*Acid  sodium  phosphate,  NaH2PO4,  can  be  prepared  by  adding 
carefully  orthophosphoric  acid  to  common  sodium  phosphate  until 
it  does  not  precipitate  barium  chlorid.  An  excess  of  the  acid  must 
be  avoided. 


84  THE   GASTRIC    JUICE. 

The  common  tests  for  the  detection  of  hydrochloric 
acid  cannot  he  employed  in  the  case  of  the  gastric  juice, 
because  the  soluble  chlorids  which  are  usually  present  will 
respond  to  them.  Special  tests  are  used,  most  of  which  are 
based  upon  the  fact  that  certain  organic  coloring  matters 
are  changed  in  color  by  a  comparatively  strong  mineral 
acid,  like  hydrochloric,  even  in  the  dilute  state,  but  are  not 
affected  by  the  weaker  organic  acids  or  acid  salts.  While 
these  methods  are  not  absolutely  accurate,  they  are  suffi- 
ciently reliable  for  clinical  purposes  when  carefully  per- 
formed. 

Many  methods  have  been  proposed  for  determining 
the  amount  of  hydrochloric  acid.  *  Sjoqvist's  method  gives 
accurate  results.  The  juice  is  evaporated  with  barium  car- 
bonate, which  contains  no  chlorids.  The  acids  unite  with 
the  carbonate,  forming  barium  salts.  The  dry  residue  is 
ignited  in  a  crucible,  when  the  salts  of  the  organic  acids 
are  destroyed,  the  barium  chlorid  remaining  unchanged. 
This  is  dissolved  with  hot  water  and  the  amount  deter- 
mined by  a  standard  solution  of  potassium  dichromate. 
This  is  added  until  the  barium  is  precipitated  and  there  is 
a  slight  excess  of  the  dichromate.  The  amount  of  dichro- 
mate used  corresponds  to  the  amount  of  barium  present, 
and  from  this  the  hydrochloric  acid  is  calculated.  Toepfer's 
method  is  not  difficult  and  the  results  are  good. 

198.  TESTS  FOR  FREE  HYDROCHLORIC  ACID. — Make 
some  0.2-per-cent.  hydrochloric  solution  as  in  Experiment 
179,  and  apply  the  following  tests: — 

a.  Add  a  methyl-violet  solution.  The  color  changes 
from  violet  to  blue. 

6.  Add  a  solution  of  tropaaolin  00.  The  yellow  color 
is  changed  to  a  red. 


GASTRIC   TESTS.  85 

c.  Add  Congo  red  solution.    The  red  changes  to  blue. 
Lactic  acid  may  produce  a  purple  color. 

Test-paper  can  be  made  from  this  reagent  by  dipping 
a  porous  paper  in  the  solution  and  drying.  It  can  be  used 
in  testing  for  HC1  like  the  solution. 

d.  Add  to  a  few  drops  of  liquid  an  equal  volume  of  an 
alcoholic  solution  containing  2  per  cent,  phloroglucin  and 
1  per  cent,  vanillin.    Evaporate  to  dryness  in  a  porcelain 
dish  on  a  water-bath  or  by  carefully  warming  over  a  flame. 
A  rose-red  color  remains.     (Gunzburg's  test.) 

e.  To  a  few  drops  of  the  HC1  solution  in  a  porcelain 
dish  add  a  little  of  an  alcoholic  solution  of  resorcin  and 
sugar.     Evaporate  to   dryness   and   a  red  color  appears. 
(Boas's  test.)     (See  page  221.) 

199.  TOEPFER'S  METHOD  FOR  DETERMINING  THE 
FREE  AND  COMBINED  HC1. — Into  each  of  three  beakers 
(A.,  B,  and  G)  measure  with  a  pipette  5  cubic  centimeters 
of  gastric  juice.  Titrate  each  of  them  with  decinormal 
ISTaOH  (4  grammes  to  the  liter),  using,  as  indicators,  in 
A  phenol-phthalein,  and  adding  the  alkali  until  the  liquid 
is  a  faint  red;  in  B  3  or  4  drops  of  a  1-per-cent.  solution 
of  alizarin  sodium  sulphonate  in  water.  The  NaOH  must 
be  added  until  the  liquid  is  a  violet  color,  not  stopping 
with  the  reddish  shade.  The  exact  shade  is  very  nearly 
that  given  by  the  indicator  to  a  1-per-cent.  solution  of  the 
common  sodium  phosphate.  With  C  the  indicator  is  3  or 
4  drops  of  a  0.5-per-cent.  alcoholic  solution  of  dimethyl- 
amido-azobenzene.  HC1  gives  a  red  color  with  this,  and 
the  NaOH  is  added  until  this  disappears  and  the  color 
changes  to  yellow. 

The  phenol-phthalein  is  turned  red  by  all  the  com- 
pounds which  have  an  alkaline  reaction.  A,  therefore,  rep- 
resents total  acidity.  The  alizarin  sodium  sulphonate  re- 


86  THE   GASTRIC   JUICE. 

acts  with  free  hydrochloric  acid,  with  organic  acids,  and 
with  acid  phosphates ;  that  is,  with  everything  except  com- 
bined hydrochloric  acid.  Consequently  A — B  represents 
the  acidity  due  to  the  hydrochloric  acid  combined  in  the 
organic  form.  Dimethyl-amido-azobenzene  is  affected  only 
by  free  hydrochloric  acid,  and  this  is  shown  in  C.  B — G 
gives  the  acidity  from  the  acid  phosphates,  plus  the  or- 
ganic acids. 

Calculate  from  the  amounts  of  sodium  hydrate  used 
the  percentages  of  each  of  these,  remembering  that  one 
cubic  centimeter  of  the  alkali  contains  0.004  gramme  and 
neutralizes  0.00364  gramme  of  HC1.  The  amounts  of  acid 
phosphates  and  organic  acids  must  be  expressed  in  terms 
of  an  equivalent  amount  of  HC1. 

200.  DETERMINATION  or  PERCENTAGE  OF  FREE  HYDRO- 
CHLORIC ACID  (SJOQVIST'S  METHOD). — To  10  cubic  centimeters  of 
filtered  gastric  juice  in  a  porcelain,  nickel,  or — better — a  platinum 
crucible  add  enough  pure  barium  carbonate  to  neutralize  the  free 
acid.  Evaporate  with  a  small  name  to  dryness;  cautiously  burn 
the  residue  and  heat  it  to  a  low,  red  heat.  After  cooling  add 
10  cubic  centimeters  of  water,  pulverize  the  residue,  and  pour  oft' 
through  a  filter  the  water  containing  the  dissolved  barium  chlorid. 
Repeat  with  successive  portions  of  water  until  the  filtrates  to- 
gether amount  to  about  50  cubic  centimeters.  Determine  in  this 
filtrate  the  amount  of  barium  chlorid  formed  from  the  hydro- 
chloric acid  in  the  following  manner:  Make  a  solution  of  10-per- 
cent, acetic  acid  and  10-per-cent.  sodium  acetate  in  water  and  add 
3  to  4  cubic  centimeters  of  this  to  the  filtrate  to  be  tested.  Add 
also  to  the  liquid  one-fourth  to  one-third  its  volume  of  alcohol. 
The  latter  is  to  make  the  barium  precipitate  more  completely,  the 
former  solution  to  prevent  the  presence  of  free  hydrochloric  acid. 
Add  from  a  burette  a  solution  of  potassium  dichromate  containing 
7.35  grammes  per  liter  until  it  is  in  excess.  This  is  shown  by 
taking  out  a  drop  from  time  to  time  on  a  glass  rod  and  putting  it 
on  a  piece  of  tetra-methyl-paraphenylene  dianiin  paper.  An  ex- 
cess of  the  dichromate  is  denoted  by  a  blue  color.  When  a  faint- 


GASTEIO   TESTS.  87 

blue  color  is  produced,  read  from  the  burette  the  amount  of  the 
dichromate  solution  used.  Calculate  from  this  the  amount  of 
acid  in  the  gastric  juice.  The  reactions  which  occur  are 

2HC1  +  BaCOs  —  BaCl2  +  CO2  +  H20 
when  the  acid  is  neutralized  by  the  carbonate,  and 

2BaCL  +  K2Cr20T  +  H20  =  2BaCr04  +  2KC1  +  2HC1 

when  the  barium  salt  is  precipitated  by  the  dichromate. 

One  molecule  of  potassium  dichromate,  weighing  294,  pre- 
cipitates two  molecules  of  barium  chlorid  containing  four  atoms 
of  chlorin.  These  four  chlorin  atoms  were  derived  from  four 
molecules  of  hydrochloric  acid  weighing  4x36.4.  Therefore,  for 
every  294  parts  by  weight  of  the  dichromate  used,  145.6  parts  by 
weight  of  hydrochloric  acid  were  present.  One  cubic  centimeter 
of  the  dichromate  solution  contains  0.00735  gramme,  and  is,  there- 
fore, equal  to  0.00364  gramme  of  hydrochloric  acid. 

The  weight  in  grammes  of  the  hydrochloric  acid  pres- 
ent in  the  gastric  juice  in  the  free  state  or  combined  with 
the  albuminous  compounds  is,  therefore,  obtained  by  multi- 
plying the  number  of  cubic  centimeters  of  dichromate  used 
by  0.00364,  and,  taking  the  weight  of  1  cubic  centimeter 
of  gastric  juice  as  1  gramme,  the  percentage  can  be  cal- 
culated. If  10  cubic  centimeters  of  juice  were  used,  it  is 
only  necessary  to  multiply  the  weight  of  HC1  in  this  by  10 
to  give  the  percentage. 

201.  VOLUMETRIC  DETERMINATION  OF  THE  FREE  ACIDS 
AND  ACID  PHOSPHATEIS. — To  10  cubic  centimeters  of  filtered 
gastric  juice1  add  5  cubic  centimeters  of  concentrated  calcium 
chlorid  solution  and  a  few  drops  of  phenol-phthalcin  as  an  indi- 
cator, then  Vio  normal  sodium  hydrate  until  it  is  neutralized, 
when  the  color  is  a  faint  pink.  The  amount  of  alkali  used  cor- 
responds to  the  total  acidity  of  the  juice.  Then  take  15  cubic  centi- 


*A  mixture  of  HC1  and  phosphates  may  be  used  if  juice 
cannot  be  obtained. 


88  THE   GASTRIC   JUICE. 

meters  more  of  the  filtered  gastric  juice  and  add  about  a  gramme 
of  finely-powdered  calcium  carbonate.  Stir  well  and  filter  through 
a  dry  paper.  Measure  10  cubic  centimeters  of  the  filtrate  into  a 
small  flask  and  by  means  of  a  rubber  bulb  or  aspirator  (not  with 
the  lungs)  blow  air  through  it  to  remove  the  carbon  dioxid.  Then 
add  5  cubic  centimeters  of  the  calcium  chlorid  solution  and  phenol- 
phthalein  and  neutralize  with  standard  sodium  hydrate  as  before. 
Since  the  free  acids  are  neutralized  by  the  calcium  carbonate,  the 
sodium  hydrate  used  in  this  second  determination  corresponds  to 
the  acid  phosphates,  and  the  difference  between  the  two  to  the 
free  acid. 

Subtract  the  number  of  cubic  centimeters  of  alkali  used  in  the 
last  determination  from  that  used  in  the  first.  If  lactic  and  volatile 
acids  are  present  and  have  been  determined,  subtract  also  the 
number  of  cubic  centimeters  required  to  neutralize  them.  The 
remainder  has  been  used  to  neutralize  the  hydrochloric  acid.  Cal- 
culate the  percentage  of  the  latter,  remembering  that  1  cubic  centi- 
meter of  sodium  hydrate  equals  0.00364  gramme  of  hydrochloric 
acid. 

Lactic  acid  changes  a  mixture  of  gentian  violet  and 
ferric  chlorid  to  a  green  or  greenish  yellow.  None  of  the 
other  normal  or  pathological  constituents  of  the  gastric 
juice  appear  to  do  the  same  or  to  interfere  with  the  use  of 
the  above  reagents  in  testing  for  the  lactic  acid. 

Lactic  acid  can  also  be  detected  in  the  gastric  juice  by 
the  yellow  color  which  it  imparts  to  a  solution  of  ferric 
chlorid  or  to  the  amethyst  solution  which  is  produced  by 
adding  ferric  chlorid  to  carbolic  acid  (phenol),  although 
glucose  or  alcohol  gives  a  similar  color. 

It  is  generally  unnecessary  to  determine  the  quantity  of 
lactic  acid.  If  this  is  desired  it  can  be  done  by  measuring  off  10 
cubic  centimeters  of  the  juice,  diluting  to  about  100  cubic  centi- 
meters and  distilling  off  the  acetic  and  butyric  acids,  which  can 
be  determined  in  the  distillate.  After  the  volatile  acids  have  been 
removed  by  distillation  until  the  liquid  remaining  is  about  10  or 
20  cubic  centimeters,  the  lactic  acid  can  be  dissolved  from  the 
residue  by  shaking  it  six  times  with  100  cubic  centimeters  of 


GASTRIC    TESTS.  89 

ether,  and  distilling  off  the  ether.  The  lactic  acid  remains.  Dis- 
solve in  water,  add  a  few  drops  of  phenolphthalein  as  an  indicator, 
and  see  how  much  decinormal  sodium  hydrate  (4  grammes  per 
liter)  is  necessary  to  neutralize  it.  Each  cubic  centimeter  of  the 
alkali  corresponds  to  0.009  gramme  of  lactic  acid. 

202.  ARNOLD'S  TEST  FOR  LACTIC  ACID. — Use  the  two 
following  solutions : —  • 

No.  1.    Saturated  alcoholic  solution  of  gentian-violet, 

0.2  cubic  centimeter. 
Distilled  water,  500.0  cubic  centimeters. 
No.  2.    Solution  of  ferric  chlorid  (U.  S.  P.),  5  cubic 

centimeters. 
Distilled  water,  20  cubic  centimeters. 

In  a  porcelain  dish  place  1  cubic  centimeter  of  No.  1 
and  add  No.  2  until  a  bluish-violet  color  results.  To  this 
add  the  filtered  gastric  juice.  Lactic  acid  changes  the  color 
to  a  green  or  greenish  yellow. 

203.*— To  about  10  cubic  centimeters  of  a  4-per-cent. 
solution  of  carbolic  acid  (phenol)  add  a  few  drops  of  a 
solution  of  ferric  chlorid.  Then  dilute  with  water  until 
the  color  is  amethyst  or  reddish-violet.  Use  this  as  a 
reagent  for  the  detection  of  lactic  acid.  The  color  is 
changed  to  yellow  by  the  lactic  acid. 

204. — Test  0.2-per-cent.  hydrochloric  acid  in  the  same 
way.  The  solution  becomes  colorless ;  that  is,  hydrochloric 
acid  gives  no  color  of  its  own,  hence  would  not  conceal  the 
lactic  acid  if  both  should  be  present.  Try  it. 

205. — Test  the  lactic  acid  with  a  solution  of  ferric 
chlorid  so  dilute  that  it  is  scarcely  colored.  The  yellow 
color  is  made  stronger. 

206. — Show  that  glucose  or  alcohol  will  give  a  yellow 
color  with  the  above  reagents.  (Experiments  203  and 
205.) 


90  THE   GASTRIC   JUICE. 

Butyric  acid  can  be  distinguished  by  its  odor,  which  is 
that  of  rancid  butter.  It  can  be  separated  from  its  solution 
by  shaking  with  ether.  The  acid  is  more  readily  soluble  in 
ether  than  in  water,  and  hence  remains  in  the  ether,  and  is 
perceptible  when  the  latter  evaporates.  It  can  also  be  re- 
moved by  distillation,  as  it  passes  off  with  the  steam.  The 
acetic  acid  is  also  volatile,  and  if  present  it  distills  with  the 
butyric  acid.  If  the  steam  is  condensed  the  quantity  of 
these  volatile  acids  can  be  determined  by  the  use  of  a  stand- 
ard solution  of  sodium  hydrate.  Since  they  both  have  the 
same  significance,  indicating  fermentation,  it  is  usually 
unnecessary  to  separate  them. 

207. — Shake  10  cubic  centimeters  of  dilute  butyric 
acid  in  a  test-tube  with  about  4  cubic  centimeters  of  ether. 
Pour  off  the  ether  and  repeat  the  operation.  Allow  the 
ether  to  evaporate,  away  from  lights  and  fires,  and  notice 
the  odor  of  the  acid  which  remains. 

The  presence  of  pepsin  in  a  solution  like  the  gastric 
juice  is  best  detected  by  trying  its  digestive  power  on  fibrin. 
Unless  the  liquid  contains  a  proper  amount  of  acid  it  must 
be  acidified  so  as  to  contain  about  0.2  per  cent,  of  hydro- 
chloric acid.  The  rapidity  of  digestion  can  be  best  per- 
ceived by  coloring  the  fibrin  dark  red  with  a  solution  of 
carmin  in  ammonia.  This  coloring  matter  is  insoluble  in 
water,  but  is  set  free,  coloring  the  liquid,  as  the  fibrin  dis- 
solves. The  depth  of  color  denotes  the  amount  digested. 
The  colored  fibrin  may  be  kept  on  hand  for  any  length  of 
time  by  pressing  out  most  of  the  water,  then  preserving  in 
glycerin  or  ether. 

208. — Stain  to  a  deep  red  some  shreds  of  washed  fibrin 
with  a  solution  of  carmin  dissolved  in  ammonia.  After 
washing  with  water  place  these  in  several  test-tubes;  add 


GASTRIC   TESTS.  91 

to  each  specimens  of  natural  or  artificial  gastric  juice,  con- 
taining different  amounts  of  pepsin,  including  one  with 
none.  If  they  are  not  sufficiently  acid  make  them  so.  Set 
the  tubes  in  a  beaker  of  water  of  about  body-temperature 
and  notice  the  setting  free  of  the  color  as  the  pepsin  acts. 

The  rennin  is  detected  by  neutralizing  and  testing 
with  milk  for  the  power  of  coagulation  as  in  Experiment 
190. 

The  test  for  starch  and  its  first  decomposition-product, 
dextrin,  is  iodin  dissolved  in  potassium  iodid.  According 
to  von  Jaksch,  neither  of  these  remains  in  the  stomach  in 
normal  digestion  after  the  first  hour,  though  they  may  be 
present  as  long  as  as  that  when  there  is  an  excess  of  acid  or 
deficiency  of  ptyalin  in  the  saliva. 

The  albuminous  substances  and  digestive  products  can 
be  detected  by  the  methods  given  in  Experiments  185,  186, 
and  187,  and  by  the  tests  described  for  albuminous  com- 
pounds. 

The  gastric  juice  never  normally  contains  blood,  but 
this  is  sometimes  found  in  it  or  in  the  vomited  material  in 
cases  of  chronic  ulceration  of  the  stomach  or  after  poison- 
ing by  the  corrosive  or  strongly  irritant  poisons.  The 
haemoglobin  is  usually  decomposed  and  the  haamatin  which 
results  gives  a  dark-brown  color,  to  the  juice.  It  is  best 
identified  by  the  hasmin  test  (Experiment  253). 

It  is  occasionally  desired  to  test  the  rapidity  of  absorption 
from  the  stomach.  This  can  be  accomplished  by  determining  how 
long  a  time  is  necessary  for  potassium  iodid  to  pass  from  the 
stomach  through  the  circulatory  system  into  the  saliva.  About 
5  grains  of  the  iodid  is  taken  in  a  capsule  or  in  water,  in  the 
latter  case  being  careful  to  thoroughly  rinse  the  mouth  afterward, 
latter  case  care  being  taken  to  thoroughly  rinse  the  mouth  after- 
ward. The  saliva  is  collected  and  tested  every  minute  after  the 


92  THE   GASTRIC   JUICE. 

fifth.  Place  a  little  upon  a  paper  dipped  in  starch-paste  and  dried, 
or  add  it  to  a  few  drops  of  starch  solution.  Then  add  a  few  drops 
of  a  solution  of  calcium  hypochlorite  (chlorinated  lime)  to  set  free 
the  iodin,  which  colors  the  starch  blue.  It  should  appear  in  the 
saliva  in  from  eight  to  fifteen  minutes.  If  the  urine  is  tested  in 
the  same  manner  there  should  normally  be  a  positive  reaction  in 
15  to  30  minutes. 

The  clinical  tests  for  hydrochloric  acid  given  above  are  open 
to  some  objections.  Many  are  interfered  with  by  large  amounts 
of  albumin  and  peptones.  If  they  fail  in  the  tests  of  a  gastric 
juice  the  biuret  test  should  be  tried,  and  if  any  of  these  substances 
are  present  they  should  be  precipitated  by  a  10-per-eent.  solution 
of  tannic  acid  and,  after  filtering,  the  liquid  should  be  again  tested 
for  hydrochloric  acid.  There  is  also  a  limit  to  the  delicacy  of  the 
tests,  so  that  very  minute  amounts  of  free  acid  may  not  be  de- 
tected. These  are  so  small  when  they  cannot  be  thus  detected  that 
the  condition  may  be  considered  pathological. 

Methyl-violet  is  turned  blue  by  about  1/3  of  a  milligramme 
of  acid.  Tropseolin  00  is  of  about  the  same  delicacy.  Congo  red 
is  somewhat  affected  by  the  organic  acids  if  they  are  not  very 
dilute.  The  test-paper  made  from  this  has  the  advantage  of  easy 
portability  and  that  it  can  be  preserved  and  also  that  something 
can  be  judged  from  the  imparted  color  of  the  amount  of  acid 
present.  With  a  large  percentage  it  becomes  a  blue  black,  and 
with  a  small  amount  a  lighter  blue.  Phloroglucin  and  vanillin 
make  a  sensitive  reagent,  it  being  possible  to  detect  with  it  one 
milligramme  of  free  acid  in  10  cubic  centimeters  of  juice. 

The  lactic  acid  test — with  phenol  and  ferric  chlorid — is  also 
in  some  cases  uncertain.  When  it  fails,  however,  the  acid  is  not 
present.  Some  common  substances,  like  sugar  and  alcohol,  give 
the  same  results  as  the  acid.  WTien  it  is  suspected  that  this  is 
the  case,  the  liquid  should  be  shaken  with  ether  to  dissolve  the 
acid,  and  the  ether,  after  separation  from  the  water,  be  evaporated 
to  dryness.  Dissolve  the  residue  in  a  little  water  and  test  it  for 
lactic  acid.  Arnold's  test  is  more  reliable. 

The  results  obtained  from  chemical  testing  of  gastric 
juice  or  vomited  material  are  often  of  great  aid  in  diagnosis. 
The  presence  of  the  organic  acids — lactic,  butyric,  or  acetic 


GASTRIC   TESTS.  93 

— more  than  thirty  minutes  after  taking  food  indicates 
fermentative  action,  usually  due  to  a  deficiency  of  hydro- 
chloric acid.  After  the  same  time  a  failure  or  excessive 
amount  of  hydrochloric  acid  can  be  considered  pathological. 
It  may  be  absent  in  acute  or  chronic  dyspepsia  and  in 
chlorosis.  It  usually  is  not  found,  or  is  present  in  only 
small  amount,  in  carcinoma  of  the  stomach.  With  dilata- 
tion of  the  stomach  caused  by  stenosis  of  the  pylorus  there 
is  often  an  hyperacidity,  more  than  0.4  per  cent,  of  the 
acid  being  present. 

Make  analyses  of  specimens  of  gastric  juice  furnished 
by  the  instructors,  handing  in  written  reports  on  the  pres- 
ence or  absence  of: — 

1.  Free  hydrochloric  acid. 

2.  Acid  phosphates. 

3.  Lactic  acid. 

4.  Butyric  acid. 

5.  Pepsin. 

6.  Coagulable  protein. 

7.  Non-coagulable  protein. 

Also  quantitative  determination  of: — 

8.  Free  hydrochloric  acid. 

9.  Combined  hydrochloric  acid. 

10.  Organic  acids  and  acid  phosphates. 
In  each  case  include  in  the  report  a  full  explanation 
of  the  results  as  showing  to  what  degree  the  juice  is  nor- 
mal or  pathological. 

THE  PANCREATIC  JUICE. 

The  fluid  secreted  by  the  pancreatic  gland  contains 
three  ferments  which  aid  in  the  digestion  of  the  food:  tryp- 
sin,  which  decomposes  the  nitrogenous  constituents; 


94  THE   PANCREATIC   JUICE. 

steapsin,  which  acts  upon  the  fats;  and  amylopsin,  which 
converts  starch  into  glucose.  The  ferments  occur  in  the 
gland  in  the  form  of  inactive  zymogens,  but  are  changed 
to  the  active  form  a  few  hours  after  death  or  by  the  action 
of  water  or  acids.  The  reaction  of  the  juice  is  alkaline  from 
the  presence  of  sodium  carbonate.  The  extract  made  from 
the  gland  by  means  of  warm  water  may  be  acid  in  reaction 
from  the  presence  of  sarco-lactic  acid. 

The  trypsin  dissolves  fibrin  and  other  albuminous  sub- 
stances, but  differs  from  pepsin  in  that  it  acts  in  a  neutral 
or  weakly  alkaline  medium.  With  trypsin  the  fibrin  does 
not  swell  and  become  transparent  before  dissolving,  as  is 
the  case  with  pepsin,  nor  is  acid  albumin  formed  as  the  first 
stage  of  digestion.  The  principal  decomposition  products 
of  fibrin  by  the  action  of  trypsin  are,  first,  a  globulin1;  then 
albumose  and  peptones;  then  leucin,  tyrosin,  asparatic 
acid,  and  other  amido  compounds.  A  substance  called 
tryptophan  (skatol-amido-acetic  acid),  which  gives  a  red- 
dish-violet precipitate  with  bromin-water  or  chlorin,  is 
produced.  The  antipeptones  are  not  affected  by  the  tryp- 
sin. The  hemipeptones  are  decomposed. 

The  substances  produced  from  albuminous  substances 
by  the  action  of  trypsin  are: — 

1.  Globulin. 

I 

2.  Albumose. 

I 

3.  Peptones  (called  amphopeptones). 

4.  Antipeptoue  5.  Hemipeptone. 

(not  further  changed). 


Leucin,  tyrosin,  aspartic  acid,  tryptophan,  etc. 

albuminous  substances  do  not  give  a  globulin  when 
acted  upon  by  trypsin;    e.g.,  serum-albumin. 


PANCREATIC   DIGESTION.  95 

Leucin  (amido-caproic  acid), 

(CH3)2CHCH2CH(NH2)COOH, 

and  tyrosin  (para-oxy-phenyl-alpha-amido-propionic  acid), 
C6H4OHCH2CH(KE2)COOH, 

are  formed  in  the  decomposition  of  protein  substances  by 
putrefaction  as  well  as  in  the  normal  processes  of  digestion, 
and  hence  they  may  be  found  as  a  result  of  pathological 
processes  where  there  is  a  destruction  of  the  proteins. 
Leucin  crystallizes  in  the  form  of  thin  plates,  which  are 
usually,  when  impure,  grouped  together  into  round  knobs 
or  balls.  These  can  be  recognized  by  the  aid  of  the  micro- 
scope. (Plate  II,  12,  &.)  In  the  impure  state  tyrosin  forms 
aggregations  of  crystals  which  resemble  those  of  leucin,  but 
when  purified  it  appears  as  fine  silky  needles  often  gathered 
into  sheaves  or  balls.  (Plate  II,  12,  c.)  Tyrosin  requires 
for  its  solution  2454  parts  of  water  at  20°.  Leucin  can  be 
dissolved  in  27  parts  of  cold  water.  This  affords  a  means 
of  separating  them. 

209. — In  a  series  of  test-tubes  place  a  small  amount 
of  pancreatic  extract,  prepared  as  in  Experiment  210.  Boil 
one  (A);  acidify  the  second  (B)  with  hydrochloric  acid; 
make  the  third  (C)  slightly  alkaline  with  sodium  carbo- 
nate; make  the  fourth  (D)  neutral  if  it  is  not  already  so. 
Place  in  each  a  shred  of  fibrin  and  set  the  tubes  in  water 
at  40°.  Let  a  fifth  tube  (E)  be  made  alkaline  and  stand 
with  fibrin  in  cold  water.  Note  the  change  in  the  appear- 
ance of  the  fibrin  and  the  speed  of  digestion.  If  the  tubes 
are  to  be  left  for  a  long  time  putrefaction  can  only  be  pre- 
vented by  the  use  of  some  antiseptic  like  chloroform  water 
(chloroform,  5;  water,  1000)  or  thymol. 

Is  solution  caused  by  an  enzyme?  What  conditions 
most  favor  it? 


96  THE   PANCREATIC    JUICE. 

210. — Prepare  an  artificial  pancreatic  juice  by  ex- 
tracting a  finely-chopped  pancreatic  gland  of  a  hog  or  ox 
with  lukewarm  water.  It  is  better  to  wait  an  hour  or  two 
after  killing  the  animal,  in  order  to  allow  of  the  forma- 
tion of  the  active  ferment,  or  to  use  0.2  per  cent,  salicylic 
acid  solution  for  the  same  purpose;  this  also  prevents 
putrefaction.  Chloroform  water  gives  an  extract  with 
marked  proteolytic  properties:  alcohol  gives  a  strongly 
lipolytic  and  amylolytic  solution.  Filter  the  extract 
through  cloth  and  add  to  the  filtrate  a  little  chloroform  or 
thymol  to  prevent  putrefaction,  which  easily  occurs  with- 
out an  antiseptic.  Place  in  the  liquid  10  to  15  grammes 
of  fibrin,  make  slightly  alkaline  with  sodium  carbonate, 
and  allow  it  to  stand  at  about  body-temperature  until  it 
has  all  dissolved.  The  products  obtained  will  depend  upon 
the  time.  If  the  digestion  is  stopped  too  soon  they  will  be 
largely  albumoses  and  peptones,  but  later  there  will  be 
more  of  the  leucin  and  tyrosin.  The  best  results  will  be 
obtained  by  digesting  for  several  hours,  or  as  much  as  a 
day. 

When  digestion  is  well  advanced  test  a  small  portion 
of  the  liquid  by  adding  bromin-water  drop  by  drop.  A 
reddish  violet  precipitate  shows  the  presence  of  tryptophan, 
which  only  appears  after  the  peptone  molecule  has  com- 
menced to  break  down.  It  is  destroyed  by  an  excess  of  the 
reagent. 

Filter  a  second  portion  and  cautiously  add  to  the  fil- 
trate acetic  acid  until  the  reaction  is  neutral.  Globulin 
or  alkali  albumin  is  precipitated.  This  may  be  coagulated 
by  boiling  and  removed  by  filtration,  then  the  filtrate  tested 
for  the  other  products.  To  this  filtrate  add  a  few  drops 
of  sulphuric  acid,  then  crystals  of  ammonium  sulphate 
until  it  is  saturated.  The  precipitate  of  albumoses  can  be 


PANCREATIC   DIGESTION".  97 

filtered  out  and  the  albumose  reactions  tried.  The  filtrate 
from  the  albumoses  contains  the  peptones.  Try  the  biuret 
test  which  should  give  a  pink  color.  On  account  of  the 
presence  of  ammonium  sulphate  a  large  excess  of  the  alkali 
must  be  employed ;  it  may  be  more  convenient  to  add  this 
in  the  solid  form. 

Let  the  remainder  of  the  original  solution  digest  for 
a  week  in  the  presence  of  thymol  or  chloroform  as  a  pre- 
ventive of  putrefaction,,  then  concentrate  the  solution  to  a 
small  bulk  and  precipitate  the  albuminous  compounds  from 
it  by  the  addition  of  about  twice  the  volume  of  alcohol. 
Filter  these  off  and  evaporate  the  alcohol.,  or  distill  it  if 
there  is  a  large  quantity.  Concentrate  the  liquid  on  the 
water-bath  to  a  thin  syrup,  then  let  it  stand  until  the 
tyrosin  crystallizes  out.  Examine  the  form  of  the  crystals 
under  the  microscope.  If  there  is  enough  of  the  liquid  to 
filter,  remove  the  tyrosin  and  let  the  leucin  crystallize 
from  the  filtrate.  If  there  is  only  a  small  amount  they 
can  both  be  identified  with  the  microscope.  Sketch  these 
and  hand  in  the  results.  If  there  is  a  sufficient  quantity 
they  may  be  tested  by  the  reactions  given  in  Experiments  . 
217-222.  Eecrystallization  often  gives  better  crystals. 

Steapsin,  the  second  ferment  of  the  pancreatic  juice, 
splits  the  fats  into  glycerin  and  the  fatty  acid  with  which 
it  was  united.  In  the  process  of  digestion,  as  it  goes  on 
in  the  animal  body,  only  a  part  of  the  fat  is  thus  decom- 
posed. The  acid  which  has  been  set  free  in  this  manner 
unites  with  the  carbonate  of  sodium  which  is  present  in  the 
intestine,  forming  the  sodium  salt  (a  soap),  and  this  serves 
to  emulsify  the  rest  of  the  fats  by  surrounding  the  globules 
with  such  a  coating  that  they  are  not  able  to  unite  into  a 
large  mass.  The  sodium  carbonate  is  not  able  to  decompose 
the  fat-molecule  or  to  form  a  soap  with  the  acid  until  the 


98  THE   PANCREATIC   JUICE. 

latter  has  been  set  free  by  the  ferment,  nor  will  it  emulsify 
the  fat  if  there  is  no  fatty  acid  present,  but  a  fine  emulsion 
is  produced  after  the  decomposition,  and  the  rest  of  the 
fat  becomes  thereby  capable  of  being  absorbed  through  the 
walls  of  the  intestine. 

The  decomposition  of  the  fat-molecule  is  as  follows: — 

C3H6(C18H3602)S  +  3H20  =  801TH,.00,H  +  C3H5(OH)3. 

stearin  stearic  acid  glycerin 

The  fat-splitting  ferment  is  easily  destroyed  by  acids; 
hence  it  may  not  be  found  in  a  gland  which  has  been  kept 
until  it  has  an  acid  reaction. 

211. — Make  a  watery  infusion  of  the  pancreatic  gland, 
as  in  the  trypsin  digestion.  If  it  is  not  already  neutral 
make  it  so.  Add  to  it  in  a  test-tube  a  few  drops  of  some 
neutral  fat,  like  olive-oil,  and  let  it  stand  for  half  an  hour 
in  a  beaker  of  water  at  about  38°,  shaking  occasionally  to 
keep  the  two  liquids  well  mixed.  Then  test  it  with  a  piece 
of  blue  litmus-paper.  It  will  turn  the  paper  red  from  the 
fatty  acid  which  has  been  set  free. 

212. — Mix  10  cubic  centimeters  of  cream  or  rich,  milk  with  a 
little  powdered  blue  litmus,  shaking  well  until  an  even  blue  color 
is  obtained.  Divide  in  two  test  tubes.  To  one  add  boiled  pan- 
creatic extract  and  keep  at  a  temperature  of  38°;  to  the  other 
unboiled.  The  former  remains  blue,  the  latter  becomes  red  from 
the  cleavage  of  the  butter-fat  into  fatty  acids  and  glycerin. 

213. — Shake,  in  a  test-tube  of  water,  to  which  a  few  drops 
of  sodium  carbonate  have  been  added,  a  few  drops  of  olive-oil 
which  does  not  contain  free  acids.  (Since  the  oil  easily  becomes 
decomposed  on  standing,  it  may  be  necessary  to  remove  the  free 
acids  by  first  shaking  with  very  dilute  sodium  hydrate  solution 
and  ether.  Separate  the  ether  from  the  water,  wash  well  by 
shaking  it  a  number  of  times  with  pure  water,  and  allow  it  to 
evaporate  at  a  gentle  heat  away  from  a  fire  or  lamp.  The  alkali 
has  united  with  the  free  acid  to  form  a  soap,  and  this  has  been 


PANCREATIC   DIGESTION.  99 

washed  out  by  the  water,  leaving  the  fat  neutral.)  If  the  oil  is 
neutral  it  will  form  no  emulsion  with  the  carbonate.  Add  now 
a  drop  of  a  fatty  acid  like  oleic  acid  and  shake.  A  fine  emulsion  is 
formed  immediately. 

214. — If  a  bottle  of  rancid  oil  is  at  hand,  shake  it  with  a  weak 
solution  of  sodium  carbonate,  and  notice  that  it  contains  enough 
of  the  fre'e  acids  to  form  an  emulsion  at  once. 

The  third  ferment  of  the  pancreas,  amylopsin,  con- 
verts starch  into  maltose  as  the  ptyalin  does,  except  that 
its  action  is  more  energetic.  Thus  it  acts  upon  raw  starch, 
which  the  ptyalin  will  not  do,  or  at  most  only  slowly. 

215. — Treat  boiled  and  unboiled  starch  with  a  small 
amount  of  the  watery  solution  of  the  pancreatic  ferments 
made  as  before.  Test  for  maltose  by  Trommels  or  Feh- 
ling's  test.  Notice  that  it  is  found  with  the  boiled  starch 
in  a  few  seconds;  after  a  longer  time  with  the  unboiled. 

216. — To  prepare  leucin  and  tyrosin  in  larger  quantities,  take 
of  white  horn-shavings  2  parts  and  boil  for  twenty-four  hours  with 
13  parts  water  and  5  parts  of  concentrated  sulphuric  acid,  adding 
water  as  it  evaporates.  Dilute  with  water,  and  while  warm  neu- 
tralize with  milk  of  lime.  Filter,  boil  the  precipitate  several  times 
with  water,  and  filter  in  order  to  remove  all  the  leucin  and  tyrosin. 
Unite  these  filtrates,  and,  after  concentrating  them  by  boiling,  pre- 
cipitate the  calcium  by  the  addition  of  oxalic  acid  without  using 
an  excess.  Filter,  extract  the  precipitate  with  boiling  water, 
unite  the  filtrates,  and  evaporate  until  it  becomes  a  thin  syrup. 
The  last  of  the  evaporation  should  be  performed  on  a  water-bath, 
to  avoid  burning.  Allow  it  to  stand  and  crystallize.  Tyrosin  at 
first  crystallizes  out,  mixed  with  a  little  leucin.  Filter,  concen- 
trate the  filtrate,  and  allow  the  leucin  to  crystallize. 

Another  better  way  to  separate  the  mixed  substances  is  to 
dissolve  them  in  a  large  quantity  of  boiling  water  to  which  am- 
monia has  been  added.  To  the  boiling  solution  add  basic  lead 
acetate  solution  until  the  precipitate  formed  is  nearly  white. 
Filter,  heat  the  filtrate  to  boiling,  neutralize  with  sulphuric  acid, 
and  filter  while  hot.  After  cooling,  the  tyrosin  crystallizes  and 


100  LEUCIN   AND   TYROSIN   REATIONS. 

can  be  filtered  off,  while  the  leucin  remains  in  solution.  Remove 
the  lead  from  the  filtrate  which  contains  the  tyrosin  by  passing 
hydrogen  sulphid  through  it,  filter,  and  concentrate  the  filtrate, 
then  boil  it  with  freshly-prepared  copper  oxyhydrate.  (This  is 
made  by  precipitating  a  solution  of  copper  sulphate  with  sodium 
hydrate  and  washing  by  decantation  until  it  no  longer  has  an 
alkaline  reaction.)  Part  of  the  leucin  is  thrown  down  as  the  copper 
salt  and  a  part  remains  in  solution.  The  precipitate  can  be  re- 
moved by  filtration  and  the  leucin  obtained  in  the  pure  state  by 
removing  the  copper  by  hydrogen  sulphid  and  allowing  the  leucin 
to  crystallize  after  concentration.  From  the  deep-blue  filtrate  the 
copper  salt  can  be  obtained  by  evaporating  it  to  a  small  volume 
and  letting  it  crystallize.  It  forms  sky-blue  aggregations  of 
crystals.  If  desired,  the  copper  can  be  removed  from  these  also 
by  hydrogen  sulphid.  The  leucin  thus  obtained  is  not  as  pure  as 
the  first  portion. 

217. — Test  the  tyrosin  with  Millon's  reagent.  It  gives  a  red 
color,  showing  the  presence  in  the  molecule  of  the  group — C0H4OH. 
(Hoffman's  test.) 

218. — To  a  portion  of  tyrosin  crystals  as  large  as  half  a  pea 
add,  in  a  porcelain  dish,  a  few  drops  of  concentrated  sulphuric 
acid  and  warm  on  the  water-bath.  This  forms  tyrosin  sulphuric 
acid.  This  is  diluted  with  water  and  enough  barium  carbonate 
added  to  neutralize  it,  then  filtered.  The  filtrate  contains  the 
tyrosin  compound,  which,  with  a  very  dilute  solution  of  ferric 
chlorid,  gives  a  deep-violet  solution  of  tyrosin  ferric  sulphate. 
(Piria's  test.) 

219. — Warm  the  tyrosin  with  a  mixture  of  formalin,  1  part; 
water,  45  parts:  concentrated  sulphuric  acid,  55  parts.  An 
emerald  green  color  results.  (Morner's  test.) 

220. — Evaporate  a  small  portion  of  the  leucin  crystals  with  a 
drop  of  nitric  acid  upon  platinum  foil.  The  residue  is  colorless, 
but  on  adding  a  drop  of  sodium  hydrate  becomes  yellow  to  brown, 
and  on  gently  heating  rolls  around  on  the  foil  in  the  form  of 
drops.  (Scherer's  test.) 

221. — Place  a  few  crystals  of  leucin  in  a  dry  test-tube  and 
heat  gently.  They  melt  with  the  odor  of  amylamin  and  sublime, 
the  leucin  appearing  on  the  sides  of  the  tube  as  wooly  flakes. 
This  may  not  succeed  if  the  leucin  is  very  impure. 


THE  BLOOET,    :r    -.  ^  ^  ;  ;  /,  101 

THE  BLOOD. 

In  the  examination  of  the  blood  it  is  convenient  to 
consider  it  as  composed  of  two  parts:  the  corpuscles  and 
the  albuminous  liquid  in  which  they  are  suspended, — the 
plasma.  The  plasma,  on  standing,  separates  into  two 
parts  by  coagulation,  the  clot — or  fibrin — and  a  liquid, — 
the  serum. 

The  reaction  of  the  blood  is  alkaline  from  the  pres- 
ence of  the  carbonate  and  phosphate  of  sodium.  The 
specific  gravity  varies  from  1.045  to  1.075,  with  an  aver- 
age for  adult  human  beings  of  about  1.055. 

The  color  of  the  blood  is  caused  by  the  red  corpus- 
cles (erythrocytes).  Even  comparatively  thin  layers  of 
the  blood  are  opaque  from  their  presence.  The  coloring 
matter  (ha3inoglobin)  can  be  set  free  from  the  corpuscle 
by  water  or  by  many  chemical  reagents.  The  color  be- 
comes then  much  darker,  since  the  light  is  no  longer  re- 
flected from  the  surface  of  the  corpuscles.  This  process 
is  called  laking.  The  addition  of  strong  neutral  salt  solu- 
tions to  blood  turns  it  bright  red,  because  of  the  increased 
reflection  of  light  from  the  shriveled  corpuscles. 

The  red  corpuscles  of  the  same  species  of  animals 
have  the  same  shape.  The  average  size  of  those  of  one 
animal  of  a  species  will  be  the  same  as  that  of  any  other, 
although  the  size  of  the  individual  corpuscle  may  vary 
greatly  in  the  same  animal.  In  most  mammals  they  are 
round,  biconcave,  non-nucleated  disks.  In  the  blood  of 
birds,  amphibians,  and  most  fishes  they  are  nucleated  and 
more  or  less  elliptical.  A  single  corpuscle  when  seen  un- 
der the  microscope  has  a  yellowish  color,  not  a  red.  The 
size  of  the  red  corpuscles  can  be  greatly  changed  by  add- 
ing water  or  strong  solutions  of  neutral  salts.  When 


102 


„,  oV,,:  :  TEE 'BLOOD. 


water  is  added  it  passes  in  by  diffusion,  and  the  corpus- 
cle swells  and  may  burst.  Likewise  by  diffusion  when 
they  are  placed  in  a  liquid  and  it  contains  more  salts  than 
the  blood,  water  passes  out  and  the  corpuscle  becomes 
smaller  and  shriveled  in  appearance.  In  order  to  dilute 
the  blood  without  changing  the  size  a  solution  containing 
0.5  to  0.6  per  cent,  of  sodium  chlorid  can  be  used.  Such 
a  solution  is  called  isotonic;  that  is,  its  osmotic  pressure 
is  the  same  as  that  of  the  fluid  within  the  corpuscles. 

Freshly-drawn  blood,  when  allowed  to  stand  undis- 
turbed, in  a  few  minutes  becomes  thickened  to  a  dark- 
red  gelatinous  mass.  If  the  coagulation  is  slow  the  red 
corpuscles  have  time  to  sink  and  collect  with  the  fibrin 
in  a  mass  in  the  lower  part  of  the  vessel.  The  serum  is 
squeezed  out  of  the  mass  and  surrounds  it,  above  and  at 
the  sides.  If  the  blood  is  beaten  during  the  time  of  co- 
agulation the  fibrin  does  not  separate  as  a  gelatinous 
substance,  but  in  stringy  masses,  which  have  a  high  de- 
gree of  elasticity.  The  coagulation  can  be  prevented  or 
hindered  by  cold  and  by  the  addition  of  neutral  salts, 
peptones,  and  some  other  substances. 

To  determine  the  number  of  red  corpuscles  the  apparatus  of 
Thoma-Zeiss  may  be  employed.  This  consists  of  two  pieces:  a 
pipette  for  measuring  and  diluting  the  blood  and  a  cell  for  count- 
ing the  number  with  the  aid  of  a  microscope.  The  lower  part  of 
the  pipette  is  a  graduated  capillary  tube  for  measuring  the  blood. 
Above  is  a  bulb  which,  being  filled  to  the  mark  with  the  diluting 
fluid,  dilutes  200  times  the  blood  which  was  measured  in  the  capil- 
lary tube.  The  counting-cell  when  covered  with  a  cover-glass  gives 
a  layer  of  blood  0.1  millimeter  in  depth.  On  the  bottom  of  the  cell 
are  ruled  sixteen  squares,  each  Vwo  of  a  square  millimeter  in  area. 
They  are  surrounded  by  two  rows  of  smaller  rectangles.  The 
volume  of  blood  over  each  of  these  squares,  then,  must  contain 
1/4ooo  cubic  millimeter.  If  the  number  of  corpuscles  which  are  con- 
tained in  this  V^  cubic  millimeter  is  determined,  the  number  in 


THE   RED    CORPUSCLES.  103 

any  volume  can  be  found  by  multiplication.  Several  different 
solutions  have  been  proposed  for  the  dilution  of  the  blood,  one  of 
the  most  convenient  being  a  3-per-cent.  solution  of  sodium  chlorid. 
For  clinical  purposes  the  blood  is  best  obtained  from  the  tip  of  the 
finger  on  the  lobe  of  the  ear.  For  testing  the  method  defibrinated 
blood  from  the  slaughter-house  may  be  employed.  The  dilution 
may  be  made  twice  as  great  by  filling  the  capillary  only  to  the  0.5 
mark  and  diluting  as  before.  In  this  case  the  number  of  corpuscles 
in  each  square  must  be  multiplied  by  8000  to  give  the  number  in  a 
cubic  millimeter. 

The  average  number  of  red  corpuscles  normally  present  is 
5,000,000  per  cubic  millimeter  in  the  case  of  a  man,  and  4,500,000  per 
cubic  millimeter  of  a  woman.  This  may  vary  greatly  in  disease. 

222.  DETERMINATION  OF  THE  NUMBER  OF  RED  BLOOD- 
CORPUSGLES. — Fill  the  capillary  tube  of  the  pipette  with  blood 
to  the  mark  1,  drawing  it  in  slowly  by  suction  with  the  mouth 
and  avoiding  the  presence  of  air-bubbles  in  the  tube.  Quickly 
wipe  dry  the  end  of  the  pipette  with  filter-paper  and  draw  in 
the  diluting  salt-solution  (3  per  cent.)  to  the  mark  101.  Close 
the  lower  end  of  the  pipette  witl*  the  finger,  then  compress  the 
rubber  tube  at  the  upper  end  of  the  pipette  and  shake  to  thoroughly 
mix  the  fluids.  The  small  glass  bead  in  the  bulb  aids  in  this  mix- 
ing. Allow  the  salt  solution  to  flow  out  of  the  capillary,  place  a 
drop  of  the  diluted  blood  on  the  ruled  side  and  cover  it  with  a 
cover-glass  so  that  air-bubbles  are  not  inclosed.  When  the  cor- 
puscles have  come  to  rest,  count  the  number  in  all  sixteen  squares. 
Count  those  upon  the  upper  and  left  hand  line  of  each  square  as 
belonging  to  the  square.  Use  an  objective  which  will  magnify 
100  to  200  diameters.  The  squares  should  be  taken  in  some  definite 
order  to  avoid  counting  the  corpuscles  in  the  same  one  more  than 
once.  The  average  number  of  corpuscles  multiplied  by  4000  gives 
the  number  in  a  cubic  millimeter  of  the  diluted  blood.  Multiply 
this  by  100  for  the  original  blood.  Since  a  slight  error  in  the 
average  of  each  square  would  make  a  considerable  error  when 
multiplied  by  4000,  it  is  advisable  to  repeat  the  filling  of  the  cell 
several  times  before  taking  the  average.  After  using,  the  pipette 
should  be  rinsed  out  first  with  the  diluting  salt  solution,  then  suc- 
cessively with  water,  alcohol,  and  ether,  and  finally  dried  by  blow- 
ing dry  air  through  it. 


104  THE   BLOOD. 

223.  SEPARATION  OF  THE   CORPUSCLES   FROM  THE 
SERUM. — Add  30  cubic  centimeters  of  a  saturated  solution 
of  sodium  chlorid  to  270  cubic  centimeters  of  water,  then 
mix  with  it  30  cubic  centimeters  of  blood,  which  has  been 
defibrinated  by  beating  it  while  freshly  drawn.     Pour  it 
into  a  flat-bottomed,  shallow  dish  and  allow  it  to  stand 
until  the  corpuscles  have  settled.    Decant  the  serum,  and, 
after  mixing  the  corpuscles  with  more  salt  solution  as  be- 
fore, allow  to  settle  and  decant  again.    By  this  means  the 
serum  can  be  entirely  removed.     Preserve  the  first  portion 
of  the  solution  for  testing  in  Experiments  234,  236,  and 
237.    If  any  of  the  corpuscles  have  been  destroyed  it  may 
be  reddened. 

224.  DETERMINATION  OF  THE  SPECIFIC  GRAVITY  OF 
BLOOD. — Prepare  a  mixture  of  benzol  and  chloroform,  of 
which  the  specific  gravity  when  tested  with  a  sensitive 
hydrometer  shall  be  somewhat  less  than  that   of  blood. 
Into  this  mixture  allow  a  drop  of  blood  to  fall.     Freshly- 
drawn  blood — for  example,  from  the  end  of  the  finger — is 
best,  though  the  method  can  be  demonstrated  by  the  use 
of  defibrinated  blood.    When  the  blood  has  sunk,  add  chlo- 
roform, drop  by  drop,  stirring  meanwhile,  until  the  drop 
floats  in  the  midst  of  the  liquid;   that  is,  it  has  the  same 
specific  gravity.     Then  filter  out  the  blood,  covering  the 
funnel  to  prevent  evaporation,  and  determine  the  specific 
gravity  of  the  mixture  by  means  of  a  sensitive  hydrometer. 
The  mixed  liquids  can  be  preserved  for  future  tests. 

225. — Wind  a  string  around  one  of  the  fingers  until  it 
is  congested,  then  prick  it  at  the  root  of  the  nail  with  a 
needle  or  knife,  sterilized  by  passing  through  a  flame. 
Test  the  reaction  of  the  blood,  using  a  piece  of  neutral, 
glazed  litmus-paper.  After  rinsing  off  the  blood  with  a 
little  distilled  water  the  paper  is  blue. 


THE   BLOOD.  105 

226.— To  a  little  defibrinated  blood  in  a  test-tube 
add  an  equal  volume  of  water,  and  notice  the  change  in 
color,  due  to  laking. 

227. — Show  that  the  blood  can  be  laked  by  adding 
a  few  drops  of  (a)  ether,  (b)  chloroform,  (c)  solutions  of 
bile  salts,  (d)  by  freezing  and  thawing,  (e)  by  heating  to 
65°. 

228. — To  another  portion  add  as  much  saturated  salt 
solution  and  observe  that  the  color  becomes  brighter. 

229. — Examine  each  of  these  under  a  microscope  and 
compare  the  appearance  of  the  corpuscles  with  those  of 
the  fresh  blood. 

230. — Place  in  a  test-tube  5  to  10  cubic  centimeters  of  a  cold, 
saturated  solution  of  sodium  sulphate.  Open  the  carotid  of  a 
rabbit  or  other  small  animal  and  allow  twice  as  much  blood  to 
flow  into  the  tube.  Collect  as  much  more  in  a  clean,  perfectly  dry 
tube  and  allow  both  to  stand  twenty-four  hours.  In  the  first  tube 
there  is  no  coagulation,  but  the  corpuscles  settle  toward  the  bot- 
tom. In  the  second  the  corpuscles  are  mostly  held  in  the  mass  of 
coagulated  fibrin  from  which  drops  of  serum  are  pressed  out. 

231. — Place  in  a  beaker  or  flask  3  to  4  cubic  centimeters  of  a 
4-per-cent.  solution  of  neutral  potassium  oxalate.  Insert  a 
cannula  into  the  carotid  artery  of  a  rabbit  or  cat  and  let  10 
times  the  above  volume  of  blood  flow  into  the  solution  without 
first  coming  into  contact  with  the  glass.  Let  it  stand  in  a  cool 
place  or  whirl  it  in  a  centrifuge  until  the  corpuscles  have  settled. 
The  clear  liquid  above  is  oxalate  plasma.  Pour  it  into  a  test- 
tube  and  notice  that  it  does  not  clot.  Add  a  few  drops  of  calcium 
chlorid  solution  when  a  clot  forms.  (Compare  this  with  the  ac- 
tion of  remain.) 

232. — From  the  oxalate  plasma  precipitate  the  fibrinogen 
with  an  equal  volume  of  saturated  salt  solution.  Apply  to  it  the 
protein  reactions. 

233. — Notice  that  the  serum  does  not  clot  with  calcium 
chlorid.  Why  not? 


106  THE   BLOOD. 

234. — Separate  the  serum  from  blood  by  collecting  the 
freshly-drawn  blood  in  a  shallow  vessel  and  letting  it  stand 
covered  until  it  has  coagulated  and  the  serum  is  pressed 
out  by  the  contraction  of  the  coagulated  mass. 

235. — Show  that  the  serum  contains  albumins  by  test- 
ing a  diluted  solution  by 

1.  Heat. 

2.  Biuret  test. 

3.  Xanthoproteic  test. 

236. — Separate  the  proteins  of  the  serum  by  adding 
to  a  portion  an  equal  volume  of  a  saturated  solution  of 
ammonium  sulphate.  This  precipitates  the  serum  glob- 
ulin. Filter  it  out,  dissolve  it  in  water,  and  try  the  glob- 
ulin reactions.  Into  the  nitrate  stir  enough  powdered  am- 
monium sulphate  to  completely  saturate  it.  Serum  albu- 
min falls  and  can  be  filtered  out,  dissolved  in  water,  and 
tested.  Test  the  filtrate  for  proteoses  with  acetic  acid  and 
potassium  ferrocyanid.  They  are  not  present.  Peptones 
are  also  absent. 

237. — Slightly  acidify  a  fresh  portion  of  the  serum 
and  boil  to  coagulate  the  proteins.  Filter  and  apply  Trom- 
mer's  test  to  the  filtrate.  Does  the  serum  contain  glucose  ? 

238. — Prepare  fibrin  by  beating  freshly-drawn  blood 
with  a  fork  or  a  bundle  of  switches.  When  it  has  coagu- 
lated, pour  off  the  liquid  and  preserve  it  for  further  tests. 
Wash  the  fibrin,  at  first  in  water  to  which  a  little  salt  has 
been  added,  then  in  clear  water.  Break  up  the  large  clots 
and  continue  the  washing  until  the  coloring  matter  is  re- 
moved. If  it  is  desired  to  keep  it,  it  can  be  preserved  in 
a  1-per-cent.  solution  of  corrosive  sublimate. 

239. — After  noticing  the  structure  and  elasticity  of 
fibrin  apply  the  following  tests: — 


HEMOGLOBIN   AND   ITS   DERIVATIVES.  107 

1.  Xanthoproteic. 

2.  Insolubility  in  hot  and  cold  water. 

240. — Let  a  shred  of  washed  fibrin  stand  for  an  hour 
or  two  in  a  test-tube  with  0.1  per  cent.  HC1.  It  swells  up 
and  becomes  transparent,  but  does  not  dissolve.  Warm 
at  60°  to  70°  for  several  hours;  filter  and  test  the  filtrate 
for  acid  albumin  by  neutralizing. 

241. — Dissolve  a  little  dried  blood  in  nitric  acid  with 
the  aid  of  heat.  Filter  and  test  the  filtrate  for  iron  with 
potassium  ferrocyanid,  which  produces  a  blue  color. 

HEMOGLOBIN  AND  ITS  DERIVATIVES. 

I.  HEMOGLOBIN:    Composed  of  an  albuminous  substance 

and  an  iron  compound, — haemochromogen. 

II.  OXYHEMOGLOBIN:  A  compound  of  oxygen  with  haemo- 

globin. 

III.  METHEMOGLOBIN:    Composition  same  as  oxyhaemo- 

globin.    Different  arrangement  of  the  atoms. 

IV.  HEMATIN:    The  iron  compound  united  with  albu- 

minous substance  in  oxyhsemoglobin.     Haemochro- 
mogen  plus  oxygen. 

V.  HEMIN:    A  compound  of  haematin  with  HC1,  one 

molecule  of  each. 

VI.  HEMOCHROMOGEN:   Haemoglobin  minus  its  albumin- 

ous constituent.    With  oxygen  it  gives  haematin. 

VII.  HEMATOPORPHYRIN:    Formed  by  the  removal  of 
iron  from  haematin,  haemin,  etc. 

HEMOGLOBIN. 

Haemoglobin,  sometimes  called  reduced  haemoglobin, 
is  the  coloring  matter  of  venous  blood.  It  contains  iron, 
besides  the  elements  which  enter  into  the  composition  of 
albuminous  substances.  The  constitution  of  the  mole- 


108  THE   BLOOD. 

cule  has  not  yet  been  determined,  although  the  formulae 
of  some  varieties  are  known  approximately;  but  it  is  very 
complex,  containing  a  large  number  of  atoms.  Like  the 
other  members  of  the  protein  compound  class,  it  contains 
an  albuminous  substance,  united  in  this  case  with  an  iron 
compound.  It  easily  unites  with  oxygen  from  the  air,  tak- 
ing up  one  molecule  of  oxygen  for  each  molecule  of  haemo- 
globin and  forming  the  readily-decomposable  compound, 
oxyhaemoglobin.  It  also  forms  compounds  with  carbon 
monoxid  (CO),  nitric  oxid  (NO),  and  sulphur,  all  of  which 
are  similar  to  its  oxygen  compound. 

Haemoglobin  can  be  obtained  from  oxyhaemoglobin 
by  the  removal  of  the  oxygen.  This  may  be  effected  either 
by  a  vacuum,  by  driving  it  out  by  means  of  a  gas  which 
itself  does  not  act  on  the  blood,  or  by  the  use  of  some 
chemical  reducing  agent.  It  is  obtained  in  the  crystalline 
state  with  more  difficulty  than  its  oxygen  compound.  It 
is  soluble  in  water,  giving  a  reddish-purple  solution. 

The  spectrum  of  haemoglobin  is  of  great  value  in  test- 
ing for  its  presence,  and  the  same  might  be  said  in  the  case 
of  the  haemoglobin  derivatives.  When  a  dilute  solution  of 
blood  is  held  before  the  slit  of  a  spectroscope,  the  tube  being- 
turned  toward  a  window,  the  solar  spectrum,  consisting  of 
the  seven  primary  colors  crossed  by  fine  dark  lines,  is  seen, 
and  in  addition  one  or  more  dark  bands,  which  are  due 
to  the  coloring  matters  of  the  blood.  That  of  haemoglobin 
has  one  broad  band  with  rather  indistinct  edges  lying  be- 
tween the  D  and  E  lines  of  the  solar  spectrum.  If  the 
liquid  in  the  tube  be  shaken  with  air  oxyhaemoglobin  is 
formed,  which  has  two  dark  bands.  For  clinical  purposes 
the  direct-vision  or  pocket  spectroscope  will  probably  be 
found  to  be  the  most  convenient  form  of  instrument. 
(Figs.  1  and  2  on  Plate  IV  show  the  spectra.) 


HEMOGLOBIN.  109 

A  fresh  alcoholic  solution  of  guaiacum  when  oxidized  gives 
u  blue  color.  Many  oxidizing  agents,  like  hydrogen  peroxid  and 
oil  of  turpentine  which  has  absorbed  oxygen  by  standing  for  some 
time  exposed  to  the  air,  will  not  act  on  the  guaiacum  alone,  but 
will  do  so  if  haemoglobin  or  its  compounds  are  present  to  serve  as 
a  carrier  of  oxygen.  This  is  often  used  as  a  test  for  blood,  but  is 
only  useful  in  a  negative  way,  for  protoplasm  will  give  the  same 
reaction.  It  can  consequently  be  obtained  from  any  cell,  like  pus 
or  mucus,  or  even  from  such  substances  as  the  potato.  If  the 
reaction  fails,  however,  there  can  be  no  blood  present. 

Since  haemoglobin  is  a  proteid,  containing  an  albu- 
minous substance,  it  will  give  the  general  reactions  of 
these  compounds. 

The  determination  of  the  amount  of  haemoglobin  in 
the  blood  is  made  by  comparing  the  color  of  the  diluted 
blood  with  a  solution  containing  a  known  weight  of  haemo- 
globin, or  with  some  other  colored  liquid  or  solid.  For 
making  a  standard  solution  of  haemoglobin  the  recrystal- 
lized  substance  is  used  (Experiment  257).  A  solution  of 
this  can  be  preserved  in  a  corked  bottle  or  sealed  tube  for 
an  indefinite  time.  The  strength  is  ascertained  by  evapo- 
rating to  dryness  a  given  volume,  and  weighing  the  residue 
of  haemoglobin.  This  method  gives  accurate  results. 

242.  DETERMINATION  OF  THE  AMOUNT  OF  HEMO- 
GLOBIN  IN  BLOOD. — Dilute  a  solution*  containing  a  known 
weight  of  haemoglobin  with  distilled  water  until  it  is  a  very 
light-red  color.  Dilute  in  the  same  way  the  blood  to  be 
tested  until  the  color  is  the  same.  For  comparison  of 
colors  the  two  solutions  may  be  placed  in  flat-bottomed 
tubes  of  colorless  glass  (Nessler  tubes)  or  in  small,  flat, 
glass  boxes,  the  breadth  of  which  between  the  parallel  sides 
is  not  more  than  a  centimeter.  Reckon  from  the  amount 
of  dilution  the  amount  of  haemoglobin  compared  with  the 
standard  solution,  also  the  absolute  weight  and  percentage. 


110  THE   BLOOD. 

For  clinical  purposes  a  convenient  instrument  for  the  determi- 
nation of  the  amount  of  haemoglobin  in  the  blood  is  that  of  Fleischl, 
called  an  haemometer.  It  consists  of  a  short,  vertical  cylinder  for 
holding  the  blood,  separated  by  a  partition  into  two  compartments; 
a  long  movable  wedge  of  ruby  glass  under  one  compartment  for 
a  standard  of  color,  and  a  white  surface  below  for  reflecting  the 
light  up  through  the  wedge  and  cylinder  to  the  eye  of  the  observer. 
The  amount  of  haemoglobin  is  found  by  filling  one  compartment 
of  the  cylinder  with  diluted  blood,  and  the  other,  over  the  ruby 
prism,  with  water.  The  prism  is  then  moved  until  the  depth  of 
color  is  exactly  the  same  as  that  of  the  blood,  when  the  percentage 
of  haemoglobin  compared  with  the  normal  amount  can  be  read  from 
the  scale.  The  following  is  the  process: — 

Use  for  a  light  a  lamp  or  yellow  gas-flame,  not  an  incandes- 
cent light  or  daylight.  Any  blood  may  be  used  for  practice.  In 
clinical  cases  use  that  obtained  from  the  tip  of  the  finger  by  the 
aid  of  a  lancet.  After  making  the  incision  force  out  a  drop  of 
blood  by  gentle  pressure.  Measure  off  the  required  volume  of  blood 
(6V2  cubic  millimeters)  by  filling  the  small  glass  tube,  open  at 
both  ends  and  mounted  on  a  handle,  which  accompanies  the 
haemometer.  This  is  accomplished  by  holding  it  horizontally  and 
dipping  one  end  into  the  drop.  Wipe  carefully  all  blood  from  the 
surface  of  the  tube.  This  will  be  more  readily  done  if  the  surface 
of  the  tube  is  kept  slightly  greasy  by  being  preserved  in  an  oily 
piece  of  chamois.  The  blood  must  exactly  fill  the  tube,  having 
neither  a  convex  nor  a  concave  surface  at  the  open  ends. 

The  compartment  over  the  ruby-glass  prism  is  to  be  filled 
with  distilled  water  by  means  of  a  pipette  and  the  other  one  not 
more  than  a  quarter  full.  Into  the  latter  the  open  glass  measuring 
tube  is  dipped  before  the  blood  has  coagulated,  and  the  haemoglobin 
is  dissolved  by  moving  the  tube  back  and  forth  so  as  to  wash  out 
the  blood.  Then  rinse  off  the  tube  into  the  blood  solution  by  the 
use  of  a  few  drops  of  water  from  the  pipette.  Fill  the  compart- 
ment from  a  half  to  three-fourths  full  of  water  and  stir  well  with 
a  wire  or  with  the  handle  of  the  measuring  tube.  Mix  the  water 
with  the  blood  until  no  turbidity  is  seen  and  until  it  is  evident 
that  the  fluid  in  the  angles  is  completely  incorporated  with  the  rest. 
Now  drop  water  from  the  pipette  upon  the  blood  solution  until  it, 
as  well  as  the  water  in  the  other  compartment,  comes  exactly  to 


OXYH^MOGLOBIN.  Ill 

the  top  of  the  division  between  the  compartments.  If  the  tip  of 
the  pipette  is  placed  slightly  below  the  surface  and  the  water  flows 
slowly  it  will  not  mix  with  the  solution  of  blood  below.  This  is 
advisable  in  order  to  prevent  the  possibility  of  any  of  the  haemo- 
globin's passing  over  into  the  other  compartment.  If  both  com- 
partments are  filled  to  the  top  of  the  separating  partition,  so  that 
there  is  no  meniscus  at  the  top  of  the  liquid,  they  appear,  when 
looked  at  from  above,  to  be  separated  only  by  a  narrow  black  line. 
A  little  grease  on  the  top  of  the  partition  will  help  to  prevent  a 
mixing  of  the  two  liquids.  (Some  authors  recommend  that  the 
water  be  allowed  to  rise  above  the  partition  and  a  cover  glass  be 
then  laid  on  top  to  prevent  currents.) 

Place  the  instrument  with  the  large  screw  at  the  right,  turn 
the  reflector  so  as  to  illuminate  the  solution,  shade  the  eye  from 
other  light,  and  move  the  ruby  prism  so  that  the  shades  of  red  in 
the  two  compartments  are  the  same.  The  figure  in  the  scale, 
opposite  the  middle  of  the  cylinder,  gives  the  percentage  of  haemo- 
globin as  compared  with  the  average  amount  found  in  normal 
human  blood. 

With  Dare's  hsemoglobinometer  but  a  single  drop  of  blood 
is  necessary.  This  is  drawn  by  pricking  the  skin  with  a  needle 
and  allowed  to  run  between  two  parallel  plates  of  glass  fixed  at 
a  short  distance  apart.  Comparsion  is  made  with  the  color  on 
the  margin  of  a  disk  which  can  be  rotated  by  means  of  a  milled 
screw  until  the  same  shade  is  attained.  Yellow  light  is  trans- 
mitted through  the  blood  and  colored  glass  from  a  candle  held 
opposite;  the  percentage  of  normal  haemoglobin  is  read  from  a 
scale  on  the  edge  of  the  revolving  disk.  The  operation  is  simpler 
and  more  expeditious  than  that  of  Fleischl. 


OXYH^EMOGLOBIN-. 

The  crystalline  form  of  oxyhsemoglobin  differs  when 
its  source  is  from  different  species  of  animal:  from  human 
blood  being  in  long  prisms;  from  the  squirrel,  flat,  six- 
sided  plates;  and  from  the  guinea-pig,  tetrahedra.  It 
can  be  easily  crystallized  from  the  blood  of  the  dog,  guinea- 


112  THE   BLOOD. 

pig,  and  rat,  but  with  more  difficulty  from  human  blood  or 
ox-blood.  The  color  of  the  crystals  is  a  bright  red,  and 
their  solution  is  a  much  brighter  red  than  that  of  haemo- 
globin, which,  when  pure,  approaches  a  black. 

Oxyhaemoglobin  is  formed  by  the  union  of  a  molecule 
of  oxygen  with  one  of  hemoglobin,  and  it  can  without 
difficulty  be  changed  back  into  haemoglobin.  The  molecule 
of  oxygen  is  in  this  compound  very  loosely  united.  Oxy- 
haemoglobin  may  be  also  considered  as  composed  of  an  iron 
compound,  haematin,  with  an  albuminous  substance.  It  is 
decomposed  into  these  two  substances  when  its  solution  is 
heated,  this  change  being  hastened  by  acids  or  alkalies. 
When  heated  with  glacial  acetic  acid  and  a  little  sodium 
chlorid  it  is  decomposed,  the  haematin  uniting  with  the  nas- 
cent HC1  formed  at  the  same  time  and  giving  haemin,  the 
microscopic  crystals  of  which  have  a  brown  color  and  char- 
acteristic form.  This  is  one  of  the  best  proofs  for  the 
presence  of  blood,  although  it  does  not  distinguish  between 
the  different  kinds.  No  other  known  substance  gives 
crystals  of  this  color  and  shape. 

The  spectrum  of  oxyhaemoglobin  consists  of  two  dark 
bands:  a  narrow  one  at  the  right  of  the  D  line  in  the 
yellow  and  a  broader  and  less  distinct  one  in  the  green 
at  the  left  of  the  E  line  of  the  solar  spectrum.  They  can 
be  made  to  vary  in  width  as  well  as  distinctness  by  mak- 
ing the  solution  more  or  less  dilute.  They  can  be  per- 
ceived when  it  contains  1  gramme  of  oxyhaemoglobin  in 
10  liters  of  water;  that  is,  1  part  in  10,000.  If  to  this 
solution  is  added  a  few  drops  of  ammonium  sulphid,  which 
has  a  reducing  action,  the  oxygen  is  removed  and  in  a  few 
minutes  the  one  broad  band  of  haemoglobin  is  seen  in  that 
part  of  the  spectrum  between  the  two  oxyhsemoglobin 


METH^MOGLOBIN.  113 

lines.  It  is  not  so  distinct  as  are  the  two  lines  and  the 
solution  may  have  to  be  strengthened  to  make  it  plainly 
visible.  The  j;wo  lines  reappear  upon  shaking  the  solu- 
tion with  air,  the  oxyhaemoglobin  being  formed  again. 

METH^EMOGLOBIN. 

In  its  percentage  composition  methaemoglobin  differs 
very  little,  if  any,  from  oxyhaemoglobin,  and  probably  is 
formed  by  a  rearrangement  of  the  atoms  in  the  oxyhaemo- 
globin  molecule.  It  is  produced  whenever  oxyhaemoglobin 
is  dried  in  the  air  at  ordinary  temperatures  or  when  it  is 
acted  upon  by  weak  acids.  Certain  oxidizing  agents  also 
will  convert  oxyhaemoglobin  into  metha3moglobin.  It  is 
also  found  in  transudations  and  cystic  fluids  which  con- 
tain blood;  moreover  in  the  urine  during  hgematuria  and 
haemoglobinuria,  as  well  as  in  the  blood  itself  in  certain 
cases  of  poisoning  or  after  a  large  destruction  of  blood- 
corpuscles  by  burns  of  the  skin. 

In  the  methasmoglobin  molecule  the  oxygen  is  more 
firmly  attached  than  in  oxyhaemoglobin,,  being  removable 
neither  by  a  vacuum  nor  by  another  gas.  Like  oxyhaemo- 
globin,  however,  it  is  changed  by  weak  acids  or  alkalies 
into  haematin  and  an  albuminous  substance.  Like  oxy- 
haemoglobin, too,  it  is  converted  by  reducing  agents  or  by 
putrefaction,  where  reducing  forces  are  at  work,  back  into 
hemoglobin. 

Methasmoglobin  crystallizes  in  brownish-red  needles 
or  sometimes  in  plates.  It  is  easily  soluble  in  water,  giv- 
ing a  brown  solution,  which  becomes  red  on  the  addition 
of  an  alkali.  The  spectrum  of  the  alkaline  methaemoglobin 
solution  has  two  bands  much  similar  to  those  of  oxyhaemo- 
globin, one  on  the  D  line,  the  other  near  the  E  line. 


114  THE   BLOOD. 


HEMATIN   AND   H^EMIN. 

Haematin  is  the  iron  compound  which  is  combined 
with  an  albuminous  substance  to  form  oxyhaemoglobin. 
It  is  set  free  whenever  oxyhaemoglobin  is  decomposed  by 
the  action  of  the  gastric  or  pancreatic  juice  or  by  an  acid. 
It  is  consequently  found  in  the  intestine  after  the  eating 
of  meat;  also  in  the  stomach  after  poisoning  by  a  mineral 
acid. 

The  formula  is  given  as: — 

C34H35N4Fe05  or  C32H32N4Fe04. 

It  may  be  obtained  from  haemin,  its  compound  with  hy- 
drochloric acid.  It  is  an  amorphous  substance,  dark  brown 
or  bluish  black. 

Hsemin  is  composed  of  haamatin  and  hydrochloric 
acid,  probably  one  molecule  of  each.  It  forms  micro- 
scopic crystals  which,  in  a  large  amount,  have  a  blue- 
black  color.  Under  the  microscope  they  are  brown,  rhom- 
bic prisms.  They  are  sometimes  separate,  but  two  are  often 
crossed  or  several  are  collected  in  clusters  or  rosettes. 
(Plate  I,  3.)  They  are  insoluble  in  water,  but  dissolve 
in  alkalies,  the  haBmatin  being  set  free.  They  are  often 
called  Teichmann's  crystals,  and  are  important  in  proving 
the  presence  of  blood. 

CARBONIC    OXID   HEMOGLOBIN. 

When  carbonic  oxid,  either  pure  or  mixed  with  other 
gases,  is  breathed  or  passed  through  blood,  it  unites  with 
the  hemoglobin,  forming  CO-hsemoglobin:  a  compound 
similar  to  oxyhaemoglobin.  It  is,  however,  a  more  stable 
compound,  the  oxygen  being  unable  to  drive  out  the  CO 


HEMOGLOBIN   DERIVATIVES.  115 

and  take  its  place.  Consequently  the  haemoglobin  is  ren- 
dered useless  as  a  carrier  of  oxygen,  and  cases  of  poisoning 
by  coal-gas,  which  contains  carbon  monoxid,  are  often  fol- 
lowed by  fatal  results.  The  compound  has  a  two-band 
spectrum  much  like  that  of  oxyhsemoglobin,  but  differs  in 
not  being  changed  to  hemoglobin  by  reducing  agents.  The 
crystals  are  similar  to  those  of  oxyhaBmoglobin,  but  more 
of  a  bluish  red.  When  mixed  with  strong  sodium  hydrate 
solution  blood  containing  carbonic  oxid  gives  a  red  mass, 
while  pure  blood  turns  brown,  with  a  greenish  cast. 

HEMOCHROMOGEN. 

As  the  oxyhaemoglobin  by  the  action  of  acids  or  alkalies  is 
decomposed  into  an  albuminous  substance  and  hsematin,  so  by  the 
same  agencies  haemoglobin  gives  an  albuminous  substance  and 
hsemochromogen.  The  latter  in  the  presence  of  free  oxygen  is 
converted  into  hsematin.  Hence,  as  we  should  expect,  by  the  re- 
moval of  oxygen  from  hsematin  by  the  aid  of  reducing  agents  we 
obtain  hsemochromogen.  The  spectrum  of  hsemochromogen,  in  an 
alkaline  solution,  has  two  bands  similar  to  those  of  oxyheemo- 
globin,  but  a  little  farther  toward  the  blue.  The  color  of  the 
alkaline  solution  is  a  cherry  red.  It  is  often  seen  in  alcoholic 
specimens  of  the  liver,  muscles,  etc.,  wrhich  have  stood  for  a  time 
in  alcohol. 

HEMATOPORPHYRIN. 

By  the  action  of  acids  upon  haematin  the  iron  is  removed, 
leaving  a  violet  to  red  coloring  matter — hsematoporphyrin.  It  is 
found  in  the  contents  of  the  stomach  and  intestine  after  poisoning 
with  strong  acids.  It  also  is  found  in  some  of  the  dark-colored 
urines.  It  is  insoluble  in  water,  more  soluble  in  acids,  and  easily 
so  in  alkalies. 

243. — Add  enough  blood  to  a  little  water  to  color  it 
a  bright  red.  Dissolve  a  small  crystal  of  ferrous  sulphate 
and  as  much  tartaric  acid  in  10  cubic  centimeters  of  water, 


116       ,  THE   BLOOD. 

then  enough  ammonia  to  make  it  faintly  alkaline,,  and  add 
1  drop  to  the  blood  solution.  The  oxyhaemoglobin  gives 
up  its  oxygen  to  the  iron  compound,  becoming  changed  in 
a  short  time  to  haemoglobin,  as  is  shown  by  the  dark  color. 
An  excess  of  the  reducing  solution  should  be  avoided. 

244. — Shake  the  dark  solution  of  hemoglobin  with 
air  and  notice  the  change  in  color  to  a  scarlet,  showing  the 
formation  of  oxyhsemoglobin. 

245. — Examine  a  very  dilute  solution  of  blood  with 
the  spectroscope  in  the  following  manner:  First  examine 
the  solar  spectrum  by  looking  through  the  spectroscope 
with  its  slit  directed  toward  a  window.  Close  the  slit  to 
a  very  narrow  opening,  and  focus  by  sliding  the  focusing 
tube  until  the  fine,  dark  lines  are  seen  clearly.  There  are 
hundreds  of  these  so-called  Fraunhofer  lines  in  the  spec- 
trum of  the  sun,  but  with  an  ordinary  instrument  many 
are  indistinct.  A  few  of  the  most  prominent  should  be 
noticed  andsused  to  locate  the  position  of  the  dark  bands 
in  the  spectra  of  haemoglobin  and  its  derivatives.  The 
most  noticeable  are  the  C  line  in  the  red,  the  D  line  in 
the  yellow,  the  E  and  &  not  far  apart  in  the  green  and  F 
in  the  blue.  If  there  are  fine,  black  lines  running  length- 
wise of  the  spectrum  they  are  caused  by  dust  in  the  slit. 
Next  make  solutions  of  blood  and  water  of  different  dilu- 
tions and  examine  the  spectrum  which  is  given  when  a 
test-tubeful  of  the  solution  is  held  before  the  slit  after  the 
solution  has  been  shaken  with  air  to  form  oxyhaamoglobin. 
Notice  the  position  of  the  two  bands  and  observe  that  this 
does  not  change  with  the  different  dilutions,  although  the 
bands  may  be  wider  or  more  distinct  when  the  solution  is 
concentrated. 

246. — To  the  solutions  of  oxyhsemoglobin  add  a  few 
drops  of  ammonium  sulphid,  and  after  they  have  stood  a 


OXYH^EMOGLOBIN   REACTIONS.  117 

short  time  examine  them  again  with  the  spectroscope.  No- 
tice that  when  the  oxygen  has  been  removed  by  the  ammo- 
nium sulphid  the  spectrum  has  changed  to  that  of  haemo- 
globin, which  consists  of  one  broad  band  in  the  space  be- 
tween that  formerly  occupied  by  the  two.  It  is  less  dis- 
tinct than  the  two  bands  of  the  oxyhsemoglobin  spectrum, 
but  can  be  rendered  darker  by  adding  3  or  4  drops  of  a 
40-per-cent.  solution  of  formaldehyd. 

247. — Shake  the  reduced  solutions  with  air  and  see 
that  the  two  bands  have  returned.  If  any  ammonium  sul- 
phid remains  the  oxyhaamoglobin  will  be  changed  again  to 
haemoglobin  on  standing. 

248. — Through  the  solution  of  blood  pass  a  stream  of 
illuminating  gas  for  a  few  minutes.  The  carbon  monoxid 
is  absorbed,  forming  carbonic  oxid  haemoglobin.  Examine 
it  with  the  spectroscope.  It  gives  two  dark  bands  much 
like  those  of  oxyhaemoglobin,  though  the  one  next  the  green 
is  not  as  wide  as  in  the  oxyhaemoglobin-spectrum.  Try 
now  to  reduce  the  compound  to  haemoglobin  by  means  of 
ammonium  sulphid.  The  carbonic  oxid  is  not  expelled, 
the  two  bands  remaining  unchanged. 

249. — Prepare  an  alcoholic  solution  of  guaiacum  by 
dissolving  some  of  the  gum  taken  from  the  middle  of  a 
lump.  No  blue  color  is  produced  in  the  solution  on  the 
addition  of  a  small  amount  of  old  oil  of  turpentine  or 
hydrogen  peroxid,  but  it  is  produced  upon  the  further 
addition  of  a  few  drops  of  blood. 

250. — Try  the  same  test  on  the  scrapings  from  a  po- 
tato. A  blue  color  is  produced  on  standing,  without  the 
aid  of  the  turpentine  or  any  similar  oxidizing  agent. 

251. — Add  a  few  drops  of  glacial  acetic  acid  to  diluted 
defibrinated  blood  and  warm  gently.  Haematin  gives  a 
brown  solution.  Examine  its  spectrum.  Neutralize  with 


118  THE   BLOOD. 

sodium  hydrate,  then  add  enough  to  dissolve  the  precipi- 
tate, warming  if  necessary.  Alkali  hsematin  is  formed. 
Examine  the  spectrum. 

252. — Acidify  slightly  a  solution  of  blood,  and  heat  to 
boiling.  Notice  the  coagulated  albuminous  substance  and 
the  dark-colored  haematin  coming  from  the  decomposition 
of  the  oxyhaemoglobin. 

253. — Filter  out  the  hasmatin  or  use  instead  a  drop 
of  fresh  blood  and  prepare  haemin  crystals  from  it  by  first 
drying  thoroughly  on  a  glass  slide,  then  adding  a  minute 
amount  of  sodium  chlorid.  Cover  with  a  cover  glass ;  add 
a  drop  of  glacial  acetic  acid,  which  will  flow  under  the 
cover.  Then  heat  over  a  small  flame  until  the  acid  boils. 
After  cooling  examine  under  a  microscope.  The  crystals 
sometimes  are  better  if  the  acid  is  added  and  the  slide 
heated  two  or  three  times. 

254. — To  prepare  a  large  amount  of  hsemin,  precipitate  the 
corpuscles  from,  defibrinated  blood  by  the  addition  of  a  large  excess 
of  a  salt  solution  which  contains  1  volume  of  a  saturated  salt 
solution  in  10  to  20  volumes  of  water.  After  twenty-four  hours 
pour  off  the  solution  and  rinse  the  precipitated  corpuscles  into  a 
flask  by  the  aid  of  a  small  amount  of  water.  Add  half  its  volume 
of  ether,  shake,  and  after  pouring  off  the  ether  which  removes 
most  of  the  fats  allow  the  solution  of  blood-coloring  matters  to 
evaporate  in  flat  dishes  at  ordinary  temperature  to  a  syrup.  Mix 
this  with  10  to  20  volumes  of  glacial  acetic  acid  and  heat  in  a 
flask  one  or  two  hours  on  the  water-bath.  Then  pour  into  a 
beaker,  add  several  volumes  of  water,  and  allow  to  stand  several 
days.  Wash  with  water  and  remove  the  albuminous  substances 
by  boiling  with  acetic  acid. 

255. — Saturate  2  or  3  cubic  centimeters  of  blood  with 
carbonic  oxid  by  passing  illuminating  gas  through  it.  Add 
to  it  twice  its  volume  of  sodium  hydrate  solution, — specific 
gravity,  1.3, — containing  about  27  per  cent.  ISTaOH.  Do 


CRYSTALLIZED   OXYH^MOGLOBIN.  119 

the  same  with  pure  blood,  and  spread  the  products  on  a 
piece  of  porcelain.  Notice  that  the  pure  blood  gives  a 
brown  color,  with  a  shade  of  green.  The  carbon  monoxid 
blood  has  a  bright-red  color  on  porcelain.  This  is  a  useful 
test  in  cases  of  suspected  poisoning  by  carbon  monoxid. 

256. — Prepare  crystals  of  oxyhsemoglobin  by  placing  on  a 
microscope-slide  a  drop  of  blood  (one  which  crystallizes  easily, 
like  that  of  a  dog,  rat,  or  guinea-pig)  and  cover  it  with  a  drop  of 
Canada  balsam.  Cover  the  whole  with  a  cover-glass  and  examine 
it  under  the  microscope.  The  crystals  will  form  in  a  few  minutes. 
They  can  also  be  made  by  mixing  the  drop  of  blood  with  a 
small  drop  of  water  on  the  slide  and  allowing  it  to  evaporate 
until  a  dry  ring  has  formed  around  it.  Place  a  cover-glass  over  it 
and  it  will  crystallize.  It  is  well  to  examine  several  different 
species  of  blood  if  they  can  be  obtained,  such  as  guinea-pig,  which 
gives  crystals  in  the  form  of  tetrahedra  (four-sided) ;  mouse,  giv- 
ing six-sided  plates;  cat  or  dog,  giving  four-sided  needles. 

257. — A  large  quantity  of  crystallized  oxyhsemoglobin  can 
be  prepared  by  the  following  method:  Make  a  solution  of  salt 
containing  1  volume  of  saturated  salt  solution  to  9  volumes  of 
water.  Add  10  volumes  of  this  to  1  of  defibrinated  blood,  and 
let  it  stand  a  day  or  two  in  shallow,  flat-bottomed  vessels  until 
the  corpuscles  have  settled.  Pour  off  the  clear  liquid,  rinse  the 
corpuscles  into  a  separatory  funnel  with  the  aid  of  as  small  a 
quantity  of  water  as  possible,  and  add  about  as  much  ether. 
Shake,  but  not  too  violently,  separate  the  solution  of  oxyhsemo- 
globin from  the  ether,  and  filter  the  former.  Cool  it  to  0°  and 
mix  it  with  one-fourth  its  volume  of  alcohol  which  has  been 
also  cooled  to  0°.  Let  the  mixture  stand  at  a  temperature  of 
— 2°  to  — 10°  for  several  days,  until  the  oxyhsemoglobin  has 
crystallized.  This  occurs  with  the  blood  of  dogs  and  rats  almost 
immediately,  but  that  of  the  ox  crystallizes  with  much  more  diffi- 
culty. After  crystallization  filter  off  the  crystals  in  the  cold,  and 
dry  by  pressing  between  filter-paper.  The  crystals  may  be  purified 
by  dissolving  in  a  small  amount  of  water,  cooling  and  precipitating 
in  the  same  manner,  repeating  several  times.  The  crystals  can  be 
preserved  for  a  standard  in  the  determination  of  the  quantity  of 
oxyhsemoglobin  in  blood. 


120  THE  BLOOD. 

258. — Test  a  parchment  dialyzer  or  tubing  to  see  that  it  has 
no  holes,  then  introduce  defibrinated  blood  diluted  with  an  equal 
volume  of  water.  Let  it  stand  an  hour  or  more  in  water.  Notice 
that  the  large  haemoglobin  molecules  cannot  pass  out,  but  the 
small  molecules  of  chlorids  do  so,  as  is  shown  by  adding  silver 
nitrate  to  the  outer  liquid. 

259. — To  a  solution  of  blood  in  water  add  a  small 
crystal  of  potassium  chlorate  or  potassium  ferricyanid. 
Methsemoglobin  is  formed,  the  color  of  the  solution  chang- 
ing from  a  red  to  a  brown.  Make  it  slightly  alkaline  and 
it  becomes  red.  Examine  the  spectrum  of  the  alkaline  so- 
lution. It  is  much  like  that  of  oxyhaBmoglobin,  though 
the  first  band  is  broader  and  extends  somewhat  farther 
toward  the  red.  It  is  reduced  to  haemoglobin  by  ammo- 
nium sulphid,  like  oxyha3moglobin. 

260. — Large  amounts  of  crystalline  methaemoglobin  can  be 
obtained  by  adding  to  a  concentrated  solution  of  oxyhaemoglobin 
enough  of  a  concentrated  solution  of  potassium  ferricyanid  to  give 
it  a  deep-brown  color.  Crystallize,  as  in  the  case  of  oxyhaemo- 
globin, by  cooling  to  zero  and  adding  one-fourth  the  volume  of 
cold  alcohol. 

261. — PREPARATION  OF  ELEMATOPORPHYRIN. — Saturate  75 
grammes  of  glacial  acetic  acid  with  hydrobromic  acid  at  10°  and 
add  to  it  in  a  300  cubic  centimeter  flask  5  grammes  of  dry  hsemin 
crystals.  Heat  thirty  minutes  on  a  water-bath  until  no  more 
hydrobromic  acid  fumes  escape,  then  pour  into  a  liter  of  water. 
After  standing  several  hours  filter  and  to  the  red  filtrate  add 
sodium  hydrate  till  the  liquid  is  neutral.  The  coloring  matter  is 
precipitated  and  should  be  filtered  off,  washed,  and  drained  upon 
filter-paper.  Digest  the  moist  precipitate  with  dilute  sodium 
hydrate  on  the  water-bath;  filter  off  the  hydrate  of  iron,  which 
separates,  and  allow  the  solution  to  stand  until  the  sodium 
compound  of  hsematoporphyrin  has  separated  in  crystalline 
masses.  Dissolve  these  in  water  and  precipitate  the  coloring 
matter  by  acidifying  with  acetic  acid.  Filter,  wash  with  water, 
and  after  stirring  the  precipitate  up  to  a  paste  with  a  small 


IDENTIFICATION   OF   BLOOD-STAINS.  121 

quantity  of  water  dissolve  by  adding  hydrochloric  acid  carefully. 
Evaporate  the  dark-red  solution  in  a  vacuum  over  sulphuric  acid. 
The  hydrochloric  acid  compound  of  haematoporphyrin  crystallizes 
in  brownish-red  needles. 

262. — Examine  the  spectrum  of  the  hsematoporphyrin. 

263. — Dissolve  a  few  haemin  crystals  in  water  by  the  aid  of 
sodium  hydrate  and  examine  the  spectrum. 

264. — Convert  the  haemin  into  haemochromogen  by  removing 
the  oxygen.  Use  for  this  purpose  a  solution  of  ferrous  sulphate 
with  tartaric  acid,  the  whole  being  made  alkaline  with  ammonia 
(Experiment  243).  Examine  the  spectrum. 

265. — If  hsemin  crystals  are  not  at  hand,  prepare  the  haemo- 
chromogen from  a  dilute  solution  of  blood  by  first  making  it 
alkaline  with  sodium  hydrate,  then  reducing  the  haematin  thus 
formed  by  ammonium  sulphid  or  ammoniacal  ferrous  tartrate. 


TESTING  SUSPECTED  STAINS  FOR  BLOOD. 

The  following  tests  can  be  used,  first  on  a  stain  made 
by  drying  a  drop  of  blood  on  a  piece  of  cloth,  then  upon 
unknown  stains. 

1.  Soak  a  small  portion  of  the  stain  in  a  few  drops 
of  water  on  a  microscopic  slide.     If  no  color  is  imparted 
to  the  liquid,  blood  is  not  present  or  the  hemoglobin  has 
been  so  much  decomposed  that  only  the  hsemin  test  will 
show  its  presence,  or,  possibly,  the  albumin  and  globulin 
have  been  coagulated. 

2.  If  a  red  color  was  seen  in  the  water  try  the  spec- 
troscopic   test.      By   drying,   both   haemoglobin   and   oxy- 
haemoglobin  may  be  changed  into  methsemoglobin,  which 
gives  a  somewhat  different  spectrum.     (Plate  IV,  11.) 

Ammonium  sulphid  reduces  this  to  hemoglobin  and 
shaking  with  air  gives  the  spectrum  of  oxyhsemoglobin 
(Experiments  246  and  247).  If  these  are  all  obtained  the 
Dresence  of  blood  is  proved. 


122  THE   BILE. 

3.  To  confirm  the  results,  or,  if  the  stain  is  too  small 
to  obtain  them,  try  to  obtain  the  haemin  crystals  as  in 
Experiment  253.     After  applying  the  acid  to  the  dried 
mass  the  latter  should  be  broken  up  with  a  glass  rod  to 
insure  thorough  mixture.    If  it  is  very  hard  let  it  soak  in 
the  acid  for  a  short  time.    These  haemin  crystals  are  pro- 
duced only  from  the  coloring  matters  of  the  blood.    When 
in  doubt  as  to  their  identity  they  should  be  compared  with 
those  obtained  from  known  blood,  remembering  that  dif- 
ferent specimens  of  crystals  may  differ  considerably  in 
size. 

4.  If  there  is  a  sufficient  amount  of  the  blood,  not 
too  long  exposed  to  the  air,  or  if  it  is  desirable  to  de- 
termine the  species  of  animal,  it  may  be  soaked  in  a  small 
amount  of  1/2-per-cent.-salt  solution  and  the  appearance 
and  size  of  the  corpuscles  compared  with  those  of  known 
specimens  or  measured  by  a  microscope  with  a  micrometer 
eye-piece. 

THE  BILE. 

The  bile  is  normally  a  brown  to  greenish,  viscid  fluid 
with  a  bitter  taste  and  a  neutral  or  slightly  alkaline  re- 
action. It  is  a  mixture  of  the  secretions  of  the  liver-cells 
with  that  from  the  mucous  membrane  of  the  passages, 
which  latter  contains  a  viscous  substance  similar  to  the 
nucleoalbumins.  This  is  usually  called  biliary  mucin,  al- 
though it  differs  in  some  respects  from  true  mucin. 

The  compounds  which  make  up  the  larger  part  of 
the  solid  matters  of  the  bile  are  the  sodium  salts  of  gly- 
cocholic  and  taurocholic  acids.  Besides  these  and  the 
biliary  mucin  there  are  present  fats,  soaps,  lecithin,  and 
cholesterin,  also  a  number  of  inorganic  salts  of  the  alka- 


THE   BILE.  123 

lies,  alkaline  earths,  and  iron.     The  color  of  the  bile  is 
due  to  the  biliary  pigments,  bilirubin,  biliverdin,  etc. 

The  salts  of  the  biliary  acids  in  the  bile  of  different 
animals  vary  in  their  proportions.  In  the  case  of  car- 
nivorous animals  only  the  taurocholic  acid  is  found;  in 
the  human  bile,  as  well  as  that  of  most  cattle,  both  are 
present.  The  biliary  acids  are  both  compounds  of  cholic 
acid,  C24H4005.  Glycocholic  acid,  C26H43N06,  is  com- 
posed of  cholalic  acid  united  with  glycocol,  CH2ISrH2C02H; 
taurocholic  acid,  C26H45]SrS07,  of  cholalic  acid  and  taurin, 
C2H4NH2S03H.  They  can  be  decomposed  into  their  con- 
stituents by  the  caustic  alkalies.  With  cane-sugar  and 
sulphuric  acid  the  biliary  acids  give  a  purple  color,  and 
this  can  be  used  as  a  test  of  their  presence. 

This  test  is  an  extremely  delicate  one,  and  its  failure  indi- 
cates that  biliary  acids  are  absent.  There  are,  however,  other 
substances — like  albumin,  morphine,  and  amyl-alcohol — which  give 
a  similar  color.  In  these  cases  the  spectroscopic  test  should  not  be 
neglected.  The  biliary  acids  can  be  obtained  pure  by  evaporating 
the  solution  to  dryness,  extracting  with  absolute  alcohol,  pre- 
cipitating this  solution  with  ether,  and  applying  the  test  to  the 
precipitate.  The  purple  solution,  when  sufficiently  diluted  with 
alcohol  and  examined  spectroscopically,  gives  a  dark  band  between 
D  and  E,  near  to  E,  and  another  before  F.  These  are  not  seen 
with  albumin,  etc. 

In  concentrated  sulphuric  acid  they  give  a  green  color, 
showing  a  strong  fluorescence.  The  sodium  salts  are  ob- 
tainable from  the  bile  by  evaporating  to  dryness  and,  after 
dissolving  in  alcohol,  precipitating  with  ether. 

Cholesterin,  C26H43OH,  occurs  in  most  of  the  fluids 
of  the  body,  as  well  as  in  the  bile,  and  the  calculi  or 
concretions  of  the  gall-bladder,  of  which  it  forms  the 
principal  part.  It  is  not  common  in  the  urine,  but  is  a 
constant  ingredient  of  the  faeces.  It  is  insoluble  in  water, 


124  THE   BILE. 

but  soluble  in  ether,  chloroform,  or  hot  alcohol.  It  crys- 
tallizes from  ether  in  fine,  silky  needles;  from  alcohol  in 
large  plates  containing  a  molecule  of  water  of  crystalliza- 
tion. (Plate  I,  4.)  In  large  quantities  it  has  the  appear- 
ance of  a  mass  of  white  plates  with  a  pearly  luster  and  a 
greasy  feeling.  It  is  distinguished  from  the  fats  by  its 
insolubility  in  the  caustic  alkalies,  even  when  boiling.  It 
forms  compounds  with  the  fatty  acids  similar  to  the  fats, 
the  cholesterin  taking  the  place  of  the  glycerin.  Lanolin, 
which  is  found  in  wool-fat,  is  an  example.  These  are  not 
easily  decomposed  by  bacteria,  hence  can  be  advanta- 
geously substituted  for  the  animal  fats  where  decomposition 
is  objectionable.  The  cholesterin  as  found  in  the  animal 
body  seems  rather  to  be  an  excrementitious  material  than 
to  have  any  function  of  its  own. 

The  bile  contains  at  least  two  well-characterized  pig- 
ments or  coloring  matters:  bilirubin,  C32H36N40G;  and 
biliverdin,  C32H36N408.  The  different  colors  of  bile  from 
a  brown  to  a  green  are  due  to  a  preponderance  of  one  or 
other  of  these.  They  seem  to  be  formed  from  the  blood- 
coloring  matters,  being  found  in  old  blood-extravasations 
and  being  increased  in  amount  in  the  bile  when  the  blood- 
corpuscles  are  destroyed,  so  that  the  coloring  matters  are 
set  free  in  the  plasma. 

Bilirubin  occurs  in  many  biliary  calculi,  particularly 
in  and  around  the  nucleus.  This  is  the  best  source  of  the 
pure  substance.  It  is  commonly  an  amorphous  powder, 
orange-red  in  color.  It  is  insoluble  in  water,  but  can  be 
dissolved  in  chloroform,  and  crystallizes  from  the  latter  in 
plates  and  prisms.  It  unites  with  strong  bases  and  in  cal- 
culi occurs  in  union  with  calcium.  By  reduction  hydro- 
bilirubin,  C32H40N407,  is  formed.  This  change  takes  place 
in  the  large  intestine  as  a  result  of  putrefactive  action,  and 


BILE   PIGMENTS.  125 

the  color  of  the  fasces  is  due  principally  to  the  hydrobili- 
rubin.  Bilirubin  is  acted  upon  by  oxidizing  agents,  with 
the  formation  of  biliverdin.  This  change  takes  place  when 
an  alkaline  solution  is  left  exposed  to  the  air. 

Biliverdin  is  an  amorphous,  green  powder.  It  differs 
from  bilirubin  in  being  insoluble  in  chloroform,  and  the 
two  can  consequently  be  separated  by  this  reagent. 

Both  of  these  biliary  pigments  when  acted  upon  by 
yellow  nitric  acid,  such  as  is  formed  by  allowing  the  strong 
acid  to  stand  in  a  bright  light,  undergo  a  change  of  color 
through  green,  blue,  violet,  and  red  to  yellow. 

Besides  these  two  coloring  matters  a  number  of  others 
have  been  described  by  different  authors.  Of  them  com- 
paratively little  is  known.  They  appear  to  be  derived  from 
biliverdin  and  bilirubin,  and  it  is  to  their  formation  that 
the  play  of  colors  is  due  when  bile  is  acted  upon  by  oxid- 
izing agents.  Some  of  these  are: — 

Biliprasin,  greenish  black. 

Bilifuscin,  brown. 

Bilicyanin,  blue. 

Choletelin,  yellow  to  brown. 

Not  infrequently  there  are  found  in  the  gall-bladder  con- 
cretions, commonly  known  as  gall-stones.  They  are  sometimes 
nearly  as  large  as  a  hen's  egg,  and  may  fill  the  bladder  almost  com- 
pletely. They  are  soft  and  often  worn  away  from  rubbing  against 
one  another.  If  they  are  cut  through  the  nucleus  is  generally 
found  to  be  dark  colored  and  composed  of  bilirubin- calcium. 
Around  this  are  concentric  layers,  usually  of  eholesterin,  but  some- 
times of  the  bilirubin-calcium.  Calcium  carbonate  is  also  found  in 
the  concretions,  as  well  as  others  of  the  biliary  pigments  in  smaller 
amounts. 

266.  SEPARATION  or  THE  SALTS  or  THE  BILIARY 
ACIDS. — The  contents  of  the  gall-bladder  of  an  ox  should 
be  mixed  with  washed  sand  and  evaporated  to  dryness  on 


126  THE   BILE. 

the  water-bath.  Then  pulverize  the  mass  in  a  mortar, 
which  operation  is  facilitated  by  the  sand.  Dissolve  the 
biliary  salts  by  strong  alcohol,,  and  filter.  Evaporate  this 
solution  to  a  small  volume  on  the  water-bath,  allow  it  to 
cool,  pour  it  into  a  flask,  and  precipitate  with  an  excess 
of  ether,  shaking  to  mix  thoroughly.  After  standing  a  few 
hours  the  precipitated  mass  is  converted  into  clusters  of 
silky  crystals  (sometimes  called  crystallized  bile).  These 
are  a  mixture  of  the  sodium  salts  of  the  taurocholic  and 
glycocholic  acids.  They  can  be  purified  by  filtering,  wash- 
ing with  water,  dissolving  in  the  smallest  possible  quantity 
of  water,  and  precipitating  again  with  ether.  They  crys- 
tallize then  in  long,  thin,  colorless  crystals  with  a  silky 
luster. 

267.  PETTENKOFER'S  TEST  FOR  THE  BILIARY  ACIDS. 
— Use  the  salts  or  the  ox-bile.  Mix  in  a  porcelain  dish  or 
test-tube  with  a  small  amount  of  concentrated  sulphuric 
acid,  being  careful  not  to  let  the  temperature  rise  above 
70°  C.  It  must,  however,  be  above  50°.  Then  add,  drop 
by  drop,  a  10-per-cent.  solution  of  cane-sugar,  stirring  with 
a  glass  rod.  A  red  color  appears.  If  too  much  sugar  is 
added  or  the  temperature  is  too  high  the  sugar  is  decom- 
posed by  the  acid,  giving  dark-brown  products,  which  con- 
ceal the  red  color. 

268. — The  same  result  may  be  obtained  by  adding  the 
sugar  to  the  liquid  to  be  tested,  acidifying  with  dilute  sul- 
phuric acid,  and  dipping  into  it  a  piece  of  filter-paper. 
Allow  the  paper  to  dry,  or  dry  it  at  a  moderate  heat,  to 
avoid  charring.  When  it  is  completely  dry,  the  red  color 
appears  on  the  paper.  If  heated  too  highly  it  will  be 
turned  black  by  the  acid. 


BILIARY   ACIDS.  127 

269. — Examine  the  spectrum  of  the  colored  liquid  obtained  in 
267.  There  are  two  absorption-bands:  one  at  F,  the  other  be- 
tween D  and  E,  near  E. 

270.  PREPARATION  OF  THE  FREE  BILIARY  ACIDS. — Dissolve 
in  water  some  of  the  sodium  salts  obtained  in  266.      Add  dilute 
sulphuric   acid   slowly,   until   a    precipitate   commences   to  form, 
then  add  a  little  ether.     After  standing  in  the  cold  until  the  acid 
has  separated,  filter,  wash,  and  recrystallize  from  a  small  amount 
of  hot  water.     This  gives  the  glycocholic  acid.     Examine  with  the 
microscope  and  sketch. 

271.  PREPARATION   OF    CHOLALIC  ACID. — Add    to   ox-bile 
one-fifth  of  its  weight  of  30-per-cent.  sodium  hydrate  and  boil 
for  twenty-four  hours,  adding  more  water  to  replace  that  which 
has   evaporated.      Then  saturate  the  liquid  with  carbon  dioxid, 
evaporate  to  dryness,  and  extract  the  mass  with  strong  alcohol. 
The  sodium  salt  of  cholalic  acid  dissolves,  as  well  as  some  other 
sodium  compounds.     Dilute  with  water  until  the  solution  contains 
no   more    than   20    per    cent,    of   alcohol,    then    precipitate   with 
dilute  barium  chlorid  as  long  as  there  is  a  precipitate.      Filter 
and  test  the  filtrate  with  barium  chlorid,  which  must  give  no  pre- 
cipitate.    Then  precipitate  the  cholalic  acid  from  this  filtrate  by 
decomposing  its  sodium  salt  by  means  of  hydrochloric  acid.     Let 
it  stand  several  hours  until  it  has  become  crystalline,  then  re- 
crystallize  from  alcohol.    Make  sketches  of  the  crystals. 

272. — Test  the  cholalic  acid  thus  obtained  with  concentrated 
sulphuric  acid,  and  notice  that  it  gives  a  green  fluorescence.  Add 
a  few  drops  of  a  cane-sugar  solution  and  see  that  a  red  color 
appears,  as  with  the  undecomposed  biliary  acids. 

273.  PREPARATION  OF  TAUROCHOLIC  ACID. — The  bile  of 
dogs  is  preferable  to  ox-bile  for  this  purpose.  It  contains  the 
sodium  salt  of  the  acid.  Evaporate  the  bile  to  dryness  on  a 
water-bath,  dissolve  in  alcohol  and  precipitate  with  ether  as  in 
266.  Dissolve  the  precipitate  in  water  and  precipitate  the  glyco- 
cholic acid  by  the  repeated  additions  of  small  amounts  of  ferric 
chlorid,  each  time  nearly  neutralizing  the  acid  reaction  with 
sodium  hydrate.  Filter  and  precipitate  the  iron  from  the  filtrate 
by  an  excess  of  sodium  carbonate.  Nearly  neutralize  the  filtrate 
and  evaporate  to  dryness.  Dissolve  in  absolute  alcohol,  evaporate 
to  dryness  and  dissolve  in  water.  From  this  precipitate  the 


128  THE   BILE. 

sodium  taurocholate  by  saturating  the  liquid  with  sodium  chlorid. 
Filter  and  add  hydrochloric  acid  until  the  solution  contains  2 
per  cent.  If  a  precipitate  of  salts  appears  remove  it  by  nitration 
and  from  the  filtrate  precipitate  the  taurocholic  acid  with  ether. 
It  should  be  filtered  immediately  and  can  be  recrystallized  in  the 
same  manner,  being  then  obtained  in  needles  or  prisms.  Its 
preparation  is  more  difficult  than  that  of  the  glycocholic  acid. 

274. — Add  the  solution  of  the  taurocholic  acid  acidified  with 
sulphuric  acid  to  solutions  of  albumin  or  peptones  and  observe 
that  they  form  insoluble  compounds. 

275.  PBEPAEATION    OF    TATJKIN. — Mix    dog-bile    with    an 
excess  of  concentrated  hydrochloric  acid,  and  evaporate  the  liquid 
to  a  small  volume  by  boiling.      Pour  off  the  solution  from  the 
resinous  mass  of  acids  which  have  separated,  and  evaporate  this 
liquid  until  the  sodium  chlorid  has,  for  the  most  part,  crystallized 
out.      Filter,  and  evaporate  the  filtrate  to  dryness.      From  the 
residue   dissolve   the   glycocoll   with   alcohol,   then   the   insoluble 
taurin  in  the  smallest  possible  quantity  of  hot  water.    'On  cool- 
ing the  taurin  crystallizes  out  in  four-sided  prisms. 

276.  PREPARATION  or   CHOLESTERIN. — Biliary  cal- 
culi or  gall-stones  are  the  best  source  of  cholesterin.    Pow- 
der the  calculus,  remove  the  bile  by  boiling  water,  then 
dissolve  the  cholesterin  in  boiling  alcohol,  and  filter  while 
hot.     It  separates  from  the  filtrate  on  cooling.     The  in- 
soluble residue,  which  consists  largely  of  compounds  of  the 
biliary  coloring  matters,  can  be  used  for  the  preparation  of 
these.     The  cholesterin  may  be  further  purified  by  dis- 
solving it  in  an  alcoholic  solution  of  potassium  hydrate 
with  the  aid  of  heat.    After  it  separates  on  cooling,  wash 
well  with  water  on  the  filter,  then  recrystallize  from  a 
mixture  of  alcohol  and  ether. 

277. — Examine  the  crystals  under  the  microscope. 
They  are  in  the  form  of  large  rhombic  tables  or  plates. 

278. — To  a  crystal  of  cholesterin  in  a  test-tube  or 
under  the  microscope  add  a  drop  of  concentrated  sulphuric 


CHOLESTERIN.  PIGMENTS.  129 

acid,  then  a  drop  of  iodin  solution.  The  crystal  becomes 
first  violet,  then  blue,  green,  and  red. 

279. — Dissolve  a  crystal  of  cholesterin  in  a  few  drops 
of  chloroform  in  a  test-tube,  then  add  an  equal  volume  of 
concentrated  sulphuric  acid.  The  chloroform  solution  be- 
comes red,  then  cherry  red,  and  purple.  On  pouring  it 
into  a  dish  it  becomes  blue,  green,  and  finally  yellow. 

280. — Evaporate  on  a  piece  of  porcelain  a  small  crys- 
tal of  cholesterin  with  a  drop  of  concentrated  nitric  acid. 
A  yellow  stain  remains  which,  if  treated  while  warm  with 
ammonia,  gives  a  red  color.  Too  high  heating  prevents  the 
reaction. 

All  these  reactions  can  be  employed  for  the  identifica- 
tion of  cholesterin. 

281.  PREPARATION  OF  THE  BILIARY  PIGMENTS. — If 
biliary  calculi  are  not  available,  bile  may  be  used  for  ob- 
taining bilirubin,  employing  the  yellow  or  brown  in  pref- 
erence to  the  green.  Dilute  it  with  a  little  water  and  add 
a  small  amount  of  lime-water,  avoiding  an  excess.  Mix 
by  shaking.  Pass  through  the  liquid  a  stream  of  carbon 
dioxid  to  convert  any  excess  into  calcium  carbonate.  Filter 
out  the  bilirubin,  which  has  been  precipitated  as  the  cal- 
cium compound,  and  wash  it  with  water.  Suspend  the 
precipitate  in  water,  decompose  it  with  a  slight  excess  of 
hydrochloric  acid,  and  shake  it  immediately  with  a  small 
amount  of  chloroform  to  take  up  the  free  bilirubin,  other- 
wise it  will  oxidize  to  biliverdin.  Separate  the  chloroform 
solution  from  the  water  and  precipitate  the  bilirubin  from 
it  by  alcohol. 

282. — If  biliary  calculi  are  at  hand  they  may  be  used 
instead  of  the  bile.  Pulverize  the  calculi,  then  dissolve 
the  cholesterin  with  a  mixture  of  alcohol  and  ether.  The 
residue  from  the  alcoholic  extraction  in  the  preparation  of 


130  THE    BILE. 

cholesterin  may  be  used  (Experiment  276).  After  the 
cholesterin  has  been  removed  decompose  with  acid  and 
proceed  as  in  the  preceding  experiment,  281. 

283. — Convert  a  portion  of  the  bilirubin  into  bili- 
verdin  by  dissolving  in  dilute  sodium  hydrate  and  letting 
the  solution  stand  in  an  evaporating  dish.  When  it  has 
turned  green,  precipitate  with  an  excess  of  hydrochloric 
acid,  filter,  and  wash. 

284. — The  biliverdin  in  an  impure  state  can  be  obtained  from 
ox-bile  by  precipitating  the  mucin  with  several  times  its  volume 
of  alcohol,  then  precipitating  the  biliverdin  by  barium  chlorid. 
Filter,  wash  with  water,  and  alcohol,  then  decompose  with  hydro- 
chloric acid.  The  biliverdin  is  insoluble  in  the  acid.  To  remove 
the  fat  it  must  be  extracted  with  ether,  then  the  biliverdin  can 
be  dissolved  in  alcohol,  which,  after  filtering,  is  left  to  evaporate. 

285.  GMELIIST'S  TEST. — To  a  solution  of  bilirubin  in 
dilute  alkali  add  slightly  yellow.,  concentrated  nitric  acid, 
holding  the  tube  in  a  slanting  position  and  pouring  slowly 
so  that  the  acid  flows  down  under  the  bilirubin  solution. 
Notice  the  colored  rings :  green  nearest  the  top,  then  blue, 
violet,  red,  and  yellow  next  to  the  acid.     The  acid  must 
not  be  too  yellow  or  the  pigments   quickly  oxidize  and 
nothing  is  seen  but  a  yellow  color. 

286.  HUPPERT'S  TEST. — To  an  alkaline  solution  of 
bilirubin    add    lime-water    to    precipitate    the    bilirubin. 
Filter,  wash  with  water,  place  in  a  test-tube  half  full  of 
alcohol  slightly  acidified  with  sulphuric  acid,  and  boil  for 
some  time.     The  bilirubin  is  oxidized  to  biliverdin  and 
the  alcohol  becomes  colored  green  or  bluish  green. 

287. — To  a  little  of  the  bilirubin  solution  in  a  test- 
tube  add  a  very  dilute  tincture  of  iodin  so  that  it  floats  on 
top.  An  emerald-green  ring  is  seen  between  the  liquids. 


CONNECTIVE   TISSUES..  131 


CONNECTIVE  TISSUES. 

Besides  the  areolar  tissue  which  may  be  the  white, 
fibrous,  or  the  yellow,  elastic,  there  are  cartilaginous  and 
osseous  tissues. 

WHITE  FIBROUS  TISSUE. 

Nearly  pure  white  fibrous  tissue  is  found  in  the  ten- 
dons which  are  made  up  of  fibers,  matrix,  and  cells.  The 
fibers  consist  of  collagen;  the  matrix  contains  mucin;  the 
cells  contain  proteins  and  salts. 

288. — Chop  a  tendon  finely  (the  tendon  of  Achilles 
can  be  used)  and  remove  the  soluble  constituents  by  wash- 
ing with  cold  water.  Extract  the  mucin  with  lime-water 
and  filter.  Preserve  the  insoluble  residue  and  use  the  solu- 
tion for  the  mucin  tests. 

289. — Show  that  mucin  gives  the  biuret  and  Milton's 
reactions. 

290. — It  cannot  be  coagulated  by  heating  nor  does  it 
dissolve  in  dilute  acids  (distinction  from  most  other  pro- 
teins) . 

291. — It  does  not  reduce  Fehling's  solution  unless  it 
has  been  boiled  for  an  hour  or  two  with  an  acid  like  hydro- 
chloric. A  reducing  amido  compound  is  thus  prod  (iced. 
When  the  acid  solution  is  neutralized  a  precipitate  of  acid 
albumin  falls.  Hence  the  original  mucin  contained  the 
reducing  substance  united  with  a  protein. 

COLLAGEN. 

292. — For  its  preparation  use  the  tendon  from  which 
the  mucin  was  removed.  Heat  the  tendon  with  a  little 
water.  On  cooling  it  gelatinizes  and  gives  the  gelatin  re- 
actions (page  60). 


132          CARTILAGE.    BONE. 

CARTILAGE. 

293. — This  contains  collagen  mixed  with  chondrogen, 
chondroitin  sulphuric  acid  and  an  albuminoid.  It  can  be 
obtained  from  pigs'  tracheas.  Separate  the  surrounding 
tissues,,  grind  the  remainder  and  boil  with  water.  The  fil- 
tered solution  yields  a  jelly  when  it  cools  (gelatin  from 
the  hydration  of  the  collagen) . 

294. — The  solution  gives  no  reaction  for  sulphuric 
acid  with  barium  chlorid  but  does  so  after  boiling  for  some 
time  with  hydrochloric  acid,  showing  that  it  contains  an 
organic  sulphate  (page  173).,  chondroid,  in  sulphuric  acid. 

295. — The  solution  reduces  Trommer's  or  Fehl ing's 
reagent  after  boiling,  but  not  before.  It  gives  the  protein 
reactions. 

BONE. 

Bone  contains  an  organic  compound,  collagen,  and  a 
number  of  inorganic  or  mineral  substances.  These  latter 
are  the  phosphates  of  calcium  and  magnesium,  mainly  the 
former;  also  calcium  carbonate  and  small  amounts  of  cal- 
cium chlorid  and  fluorid.  The  inorganic  substances  can 
be  removed  by  acids,  leaving  the  bone  flexible.  If  a  bone 
is  heated  the  collagen  is  decomposed,  with  an  evolution  of 
ammonia,  showing  that  the  collagen  is  a  nitrogenous  com- 
pound. Then  inflammable  gases  are  set  free.  If  the  igni- 
tion is  performed  where  free  access  of  air  is  prevented, 
there  remains  a  black  mass  known  as  bone-black  or  animal 
charcoal.  The  black  color  is  due  to  carbon,  which  can  be 
removed  by  burning  in  the  air,  leaving  the  mineral  or  in- 
organic constituents  only.  The  bone-black  is  an  extremely 
porous  substance  and  has  the  power  of  absorbing  from 
their  solutions  many  of  the  vegetable  coloring  matters  and 


CONSTITUENTS   OF   BONE.  133 

also  the  alkaloids.  On  this  account  it  is  used  for  decolor- 
izing liquids  as  well  as  for  an  antidote  in  cases  of  poison- 
ing by  strychnine  and  some  other  alkaloids. 

296. — Fill  a  dry  test-tube  one-third  full  of  fragments 
of  dry  bone,  fasten  it  horizontally  by  the  upper  end  in  a 
clamp,  and  heat,  at  first  gently,  then  to  as  high  a  tem- 
perature as  possible  without  softening  the  glass,  moving 
the  burner  so  as  not  to  heat  it  in  one  spot.  The  organic 
matter  is  decomposed.  First  water  is  given  off,  then  am- 
monia. Test  this  with  a  piece  of  red  litmus-paper.  An 
oily  or  tarry  mixture  distills  off  with  inflammable  gases. 
When  the  gas  has  been  expelled  the  mineral  matters  of  the 
bone  remain  mixed  with  carbon. 

297. — Take  a  piece  of  this  and  heat  it  in  the  air,  hold- 
ing it  with  the  forceps  or  a  piece  of  wire.  The  carbon 
burns  away,  leaving  only  the  mineral  matters  as  a  brittle 
mass. 

298. — Dissolve  the  bone-ash  in  dilute  nitric  acid. 
Notice  that  carbonates  are  present,  as  shown  by  the  effer- 
vescence of  carbon  dioxid  gas. 

299. — Test  a  portion  of  the  solution  for  phosphoric 
acid,  which  is  present  in  the  phosphates,  by  making  it 
strongly  acid  with  nitric  acid,  then  adding  ammonium 
molybdate  and  warming  gently.  A  yellow  precipitate 
shows  phosphoric  acid. 

300. — Test  another  small  portion  of  the  solution  for 
chlorids  with  silver  nitrate.  They  give  a  milkiness  or  a 
white  precipitate,  but  are  present  in  very  small  quantities. 

301. — Test  the  remainder  of  the  nitric  acid  solution 
for  calcium  and  magnesium  after  removing  the  phosphoric 
acid  in  the  following  manner:  Add  a  few  drops  of  ferric 
chlorid  to  the  solution  in  a  beaker.  The  iron  unites  with 
the  phosphoric  acid,  which  was  held  by  the  calcium  and 


134  BONE. 

forms  ferric  phosphate.  Pour  a  few  drops  from  the  beaker 
into  a  test-tube  and  test  by  adding  ammonia  to  see  if  all 
the  phosphoric  acid  has  united  with  the  iron.  If  this  is 
the  case,  the  ammonia  gives  a  yellowish  precipitate.  If 
not  enough  ferric  chlorid  was  added  the  precipitate  is 
white,  and  more  of  the  iron  solution  must  be  added  and 
the  test  repeated  until  all  the  phosphoric  acid  has  been 
taken  by  the  iron.  Then  to  the  solution  add  a  solution 
of  sodium  carbonate  until  it  is  nearly  neutral;  that  is, 
until  the  precipitate — which  forms  as  the  sodium  carbonate 
strikes  the  liquid — dissolves  slowly  on  stirring.  Then 
add  1  or  2  grammes  of  barium  carbonate  to  precipitate 
the  ferric  phosphate.  Filter  after  warming.  Precipitate 
the  barium  from  the  hot  filtrate  with  dilute  sulphuric  acid 
and  filter.  Having  thus  removed  the  phosphoric  acid,  test 
the  filtrate  for  calcium  by  making  it  alkaline  with  am- 
monia and  adding  ammonium  oxalate  as  long  as  a  pre- 
cipitate is  formed.  The  calcium  is  thrown  down  as  white 
calcium  oxalate.  Filter  and  to  the  filtrate  add  sodium 
phosphate.  A  white  crystalline  precipitate  shows  mag- 
nesium. 

302. — Test  the  absorptive  power  of  bone-black  by  adding 
it  to  a  light-blue  solution  of  indigo  and  warming,  then  filtering. 
The  coloring  matter  will  have  almost  or  entirely  disappeared  from 
the  filtrate. 

Directions  have  been  given  before  (Experiment  138) 
for  the  separation  of  the  collagen  from  the  mineral  con- 
stituents of  the  bone. 

MUSCULAR  TISSUE. 

A  muscle  is  made  up  of  fibers  or  cells,  consisting  of 
a  sheath  (the  sarcolemma),  composed  of  a  substance  similar 


MUSCULAR   TISSUE.  135 

to  elastin,  and  its  contents.  The  latter  are  mostly  albu- 
minous matters,  alkaline  and  liquid  during  life,  but  be- 
coming acid  and  more  solid  after  death.  This  albuminous 
liquid,  which  in  many  respects  corresponds  to  the  plasma 
of  the  blood,  is  called  muscle-plasma.  It  coagulates  quickly 
at  the  ordinary  temperatures,  and  thus  gives  rise  to  the 
rigor  mortis  of  the  muscles  after  death.  The  coagulated 
mass  is  myosin:  a  mixture  of  a  number  of  compounds. 
The  cause  of  the  coagulation  of  the  myosin  in  this  case 
is  probably  the  formation  of  lactic  acid,  which  accumulates 
in  the  muscle  after  death. 

If  fresh  muscular  tissue  is  treated  with  boiling  water, 
most  of  the  albuminous  substances  are  coagulated  and 
upon  filtering  remain  with  the  fats  in  the  insoluble  residue. 
The  solution  contains,  besides  inorganic  matters,  a  class 
of  organic  compounds,  sometimes  called,  from  the  method 
of  obtaining  them,  the  "extractives."  They  may  be  divided 
into  two  groups:  those  which  contain  no  nitrogen  and 
those  of  which  nitrogen  is  a  constituent.  Among  the  non- 
nitrogenous  are  lactic  acid  and  its  compounds;  also  gly- 
cogen  and  its  derivatives:  dextrin,  maltose,  and  glucose. 
The  principal  ones  of  the  nitrogenous  extractives  are 
creatin  and  creatinin,  small  quantities  of  urea  and  uric 
acid,  and  the  nuclein  bases,  such  as  guanin,  xanthin,  and 
hypoxanthin — formerly  called  sarcin.  Carnin,  which  is 
similar  in  properties  and  composition  to  the  nuclein  bases, 
is  also  found  in  the  watery  extract  of  muscle. 

Creatin: 

H9N     HOCO 

I  I 

0  I 

I  I 

H3CN OH, 


136  THE   UKINE. 

and  creatinin: 

HN  — CO 


H3CN  — CH2 

are  closely  related,  the  latter  being  derived  from  the  former 
by  taking  away  one  molecule  of  water,  and  can  be  changed 
back  into  creatin  by  adding  the  water  again. 

The  nuclein  bases,  also  called  purin  bases,  xanthin 
bases  and  alloxuric  bases,  guanin  (C5H5N50),  hypoxanthin 
(C5H4N"40),  and  xanthin  (C5H4N402)  are  similar  in  their 
properties  and  related  to  uric  acid  (C5H4N403).  They 
are  derivatives  of  the  hypothetical  nucleus,  purin: 

(6) 
(1)N=       CH 

I  (7) 
(2 )  HC     (5)  C  —  NH 

II  II  >  CH  (8) 
(3)N—       C-       N 

(4)        (9) 

They  are  formed  by  the  addition  to,  or  substitution  in 
this  of  oxygen  or  organic  radicals,  the  position  being  in- 
dicated by  the  figures.  Thus  guanin  is  2-amino-6-oxy- 
purin.  Hypoxanthin  is  6-oxypurin;  xanthin,  2-6-dioxy- 
purin  and  uric  acid,  2-6-8-trioxypurin.  They  occur  partly 
free  in  the  muscular  tissue  and  partly  united  with  phos- 
phoric acid  and  albuminous  substances  in  the  form  of 
nucleins.  All  these  soluble  compounds  are  found,  natur- 
ally, in  the  various  meat-extracts,  which  are  used  in  foods. 
They  are  probably  formed  in  the  body  by  the  decompo- 
sition of  the  albuminous  materials.  When  taken  as  a  food, 


PREPARATION    OF   UREA.  137 

their  value  is  rather  in  the  stimulation  of  digestion  through 
their  agreeable  taste  than  in  their  absolute  nutritive  worth. 
This  may  be  merely  because  of  their  increasing  the  secre- 
tion of  the  digestive  fluids. 

303. — Pith  a  frog  (see  Experiment  305)  :  cut  out  the 
gastrocnemius  muscles.  Let  one  remain  in  water  at  a  tem- 
perature of  50°  until  rigor  caloris  has  appeared.  Put  the 
other  into  boiling  water.  Test  the  reaction  of  both  with 
litmus  paper.  The  former  is  acid:  the  latter  is  alkaline. 

304. — Kill  a  rabbit  or  a  frog;  at  once  lay  bare  the 
muscle  and  test  it  with  red  and  blue  litmus  paper  and  with 
lacmoid  paper.  To  the  latter  it  is  neutral;  the  red  litmus 
turns  blue  and  the  blue  litmus,  red — that  is,  the  reaction 
toward  litmus  is  amphoteric.  Both  free  acids  and  acid 
salts  turn  litmus  red ;  lacmoid  becomes  red  from  the  action 
of  free  acids,  not  from  that  of  the  acid  salts.  Let  the 
muscle  stand  and  test  it  as  before.  It  remains  neutral  to 
lacmoid,  but  is  acid  to  litmus.  Lactic  has  been  produced, 
but  instead  of  remaining  in  the  free  state  it  forms  lac- 
tates.  The  bases  are  thus  removed  in  part  from  other  salts, 
leaving  them  as  acid  salts  (for  example,  acid  phosphates), 
which  latter  produce  the  acid  reaction. 

305. — Inject  a  solution  of  acid  fuchsin  into  the  subcutaneous 
lymph-space  of  a  frog.  After  it  has  been  absorbed  pith  the  ani- 
mal; that  is,  destroy  the  brain  by  pushing  forward  a  stout  wire 
inserted  through  the  occipito-atlantoid  membrane.  This  lies  in 
the  middle  of  a  line  drawn  across  the  back  of  the  head  through 
the  posterior  margin  of  the  tympanic  membranes.  Strip  the 
skin  from  both  hind  legs  and  separate  the  muscles  of  one  thigh 
until  the  sciatic  nerve  is  exposed.  Hang  the  frog  by  a  hook 
through  the  jaws  and  repeatedly  stimulate  the  sciatic  nerve  by 
electrodes  passing  under  it.  The  stimulated  leg  contracts  vigor- 
ously while  the  other  remains  passive.  In  the  working  muscle 
lactic  acid  is  formed  and  this  decomposes  the  colorless,  alkaline 


138  MUSCULAR   TISSUE. 

salt  of  fuchsin,  producing  a  pink  or  red  color.    In  the  resting  leg 
there  is  no  such  acid  formation  and  the  color  remains  pale. 

306. — Chop  finely  20  to  25  grammes  of  lean  meat  and 
extract  it  for  an  hour  with  three  times  as  much  cold  water, 
stirring  frequently.  Filter  through  muslin  and  test  the 
filtrate  for  myogen,  an  albumin,  sometimes  called  myo- 
sinogen.  It  is  coagulable  and  gives  the  general  reactions 
for  albuminous  substances  (page  37).  It  is  soluble  in 
distilled  water  and  does  not  precipitate  from  this  solution 
on  dialysis. 

307. — Soak  the  residue  from  the  last  experiment  in 
50  to  75  cubic  centimeters  of  10  per  cent,  ammonium 
chlorid  solution,  filter  through  muslin  and  test  the  filtrate 
for  myosin,  a  globulin,  sometimes  called  paramyosinogen 
or  musculin.  It  responds  to  the  globulin  tests  (page  45) 
as  well  as  the  general  reactions  for  albuminous  substances 
(page  37)  and  is  coagulated  by  heat. 

308. — Mince  finely  10  to  15  grammes  of  fresh  muscular  tissue 
and  extract  it  by  stirring  with  water  for  a  few  minutes.  Warm 
the  filtered  solution  in  a  double  beaker  (Experiment  91).  When 
coagulation  occurs  filter  and  keep  the  temperature  constant  until 
no  further  change  is  observed,  then  increase  it.  Note  the  tempera- 
tures at  which  the  different  proteins  coagulate  and  report  them. 
This  is  the  method  of  separation  by  fractional  coagulation. 

309.  PREPARATION  OF  MUSCLE-PLASMA. — Kill  a  frog  and 
immediately  wash  the  blood  from  the  body  by  passing  in  through 
a  cannula  inserted  in  the  aorta  a  cold  0.5-per-cent.  sodium  chlorid 
solution.  The  necessary  force  can  be  gained  by  placing  the  solu- 
tion in  a  doubly-tubulated  bottle,  which  can  be  raised  and  lowered, 
and  connecting  the  lower  tubulure  with  the  cannula  by  a  small 
rubber  tube.  Cut  the  muscle  up  as  quickly  as  possible  with  a  cold 
knife  or  pair  of  scissors  and  freeze  it  by  stirring  in  a  beaker,  pre- 
viously surrounded  by  a  freezing  mixture  of  ice  (3  parts)  and  salt 
(1  part).  It  freezes  at  about  —7°  C.  Then  rub  it  to  as  fine  a 


CONSTITUENTS   OF   MUSCLE.  139 

powder  as  possible  in  a  mortar,  whir>h,  as  well  as  the  pestle,  has 
been  cooled  below  this  temperature  by  standing  in  a  freezing 
mixture.  Subject  the  mass  to  a  strong  pressure,  which  gives  a 
yellowish  liquid.  Filter  this  through  muslin  at  a  temperature 
below  freezing.  The  nitrate  is  the  muscle-plasma.  Through  the 
whole  process  care  must  be  taken  to  preserve  a  low  temperature 
to  prevent  coagulation. 

310. — Pour  a  few  drops  of  the  plasma  into  a  dish  of  the 
ordinary  temperature.  It  coagulates  immediately. 

311. — Test  the  reaction  of  the  plasma  to  litmus-paper.  It 
is  alkaline. 

312. — Allow  the  temperature  of  the  rest  of  the  plasma  to 
rise  slowly,  and  notice  that  it  coagulates  at  a  little  above  freezing. 
On  standing,  a  yellowish  liquid  is  pressed  out  of  the  clot,  as  in 
the  case  of  the  coagulum  of  blood-plasma,.  This  is  muscle-serum. 

313. — Try  the  reaction  of  the  muscle-serum  to  litmus-paper. 
It  is  alkaline. 

314. — Prove  that  the  coagulated  mass  is  a  globulin  (Experi- 
ments, 98,  99,  100,  and  101). 

315. — Take  about  500  grammes  of  lean  beef,  and,  after  re- 
moving, as  completely  as  possible,  the  fat  and  connective  tissue, 
chop  it  finely.  Add  an  equal  weight  of  water  and  heat  half  an 
hour  on  a  water-bath  to  55°  or  60°.  Filter  through  muslin,  press- 
ing out  the  water  with  the  hands.  Repeat  the  extraction  with 
half  as  much  water.  Unite  the  nitrates,  and  boil  to  precipitate 
the  albuminous  compounds.  (Instead  of  the  meat  a  jar  of  beef- 
extract  can  be  used  after  dissolving  in  water.)  Filter  and  add  lead 
acetate  as  long  as  a  precipitate  forms,  avoiding  a  great  excess. 
Filter  and  remove  the  lead  by  passing  hydrogen  sulphid  gas  into 
the  solution.  Filter  out  the  lead  sulphid  and  evaporate  the  nitrate 
on  the  water-bath  to  5  or  10  cubic  centimeters.  Allow  the  yellow- 
ish syrupy  liquid  to  stand  two  or  three  days  in  a  cool  place,  when 
the  creatin  crystals  will  separate.  Filter,  and  wash  with  88-per- 
cent, alcohol.  Unite  the  nitrate  and  the  washings  and  remove  the 
alcohol  by  evaporation  on  a  water-bath.  After  cooling  make  alka- 
line with  ammonia  and  add  an  ammoniacal  solution  of  silver 
chlorid.  Filter.  The  precipitate  contains  the  silver  compounds  of 
hypoxanthin,  xantMn,  and  guanln.  (The  nitrate  contains  lactic 
acid.  Preserve  for  testing.)  Wash  with  ammonia  and  dissolve 


140  MUSCULAR   TISSUE. 

in  boiling  HNO8,  sp.  gr.,  1.1.,  to  which  a  little  pure  urea  has  been 
added  to  prevent  the  decomposition  of  the  bases.  While  hot,  filter 
from  a  small  amount  of  AgCl,  which  may  remain,  then  allow  to 
stand  twelve  hours.  Hypoxanthin-silv&r  nitrate  separates  in  small 
needle-shaped  crystals.  Filter  and  wash  with  water.  From  the 
filtrate,  by  the  addition  of  an  excess  of  ammonia,  is  obtained  a 
slight  precipitate  of  wantMn-silver  oxid.  The  free  xanthin  and 
hypoxanthin  may  be  obtained  by  suspending  their  silver  com- 
pounds in  water  and,  after  heating  and  making  slightly  alkaline 
with  ammonia,  adding  ammonium  sulphid  drop  by  drop  until  the 
silver  is  precipitated,  avoiding  an  excess.  On  evaporating  the 
filtrate  the  xanthin  and  hypoxanthin  will  be  left  as  microscopic 
crystals. 

Most  of  the  guanin  is  left  in  the  precipitate  made  by  the  am- 
monium sulphid.  It  can  be  dissolved  by  boiling  with  a  little  very 
dilute  hydrochloric  acid.  Filter  and  precipitate  it  from  the  filtrate 
by  making  it  alkaline  with  ammonia. 

To  obtain  the  lactic  acid  from  the  filtrate  from  the  precipitated 
hypoxanthin,  etc.,  first  precipitate  the  silver  by  HjjS  and  filter. 
Concentrate  the  filtrate  on  the  water-bath  until  most  of  the  am- 
monia has  been  expelled.  Then  cool  and  acidify  strongly  with 
dilute  sulphuric  acid.  The  lactic  acid  is  thus  set  free  and  can 
now  be  separated  by  shaking  gently  with  about  one-fifth  of  its 
volume  of  ether,  which  dissolves  the  lactic  acid,  but  not  the 
sulphuric.  After  shaking  in  a  glass-stoppered  funnel,  allow  it  to 
stand  until  the  ether  has  all  risen  to  the  top  of  the  liquid.  Then 
draw  off  the  water  and  the  ether  into  separate  flasks.  Repeat  the 
operation  a  few  times  with  fresh  portions  of  ether.  Mix  the  differ- 
ent portions  of  ether  and  distill  or  evaporate  it.  The  residue  con- 
tains the  lactic  acid  mixed  with  a  little  sulphuric.  Dilute  with 
water  and  boil  a  minute  with  zinc  carbonate  until  it  has  lost  its 
acid  reaction.  Filter,  and  evaporate  the  filtrate  on  the  water-bath 
to  a  small  volume.  Then  let  it  stand,  and  the  zinc  lactate  will 
crystallize  in  four-sided  prisms:  (C8HA)2Zn  +  2H2O.  Filter 
these  from  the  remaining  liquid,  and  dry  on  filter-paper. 

To  obtain  the  free  acid  dissolve  some  of  the  crystals  in  water 
and  precipitate  the  zinc  with  hydrogen  sulphid  gas.  Filter,  and 
evaporate  the  filtrate.  The  acid  will  be  left  as  a  syrupy  liquid. 
Test  it  for  its  acid  reaction  and  sour  taste.  It  differs  from  fer- 


THE   BRAIN.  141 

mentation  lactic  acid  in  that  it  rotates  the  plane  of  polarized  light 
toward  the  right.  Fermentation  lactic  acid  does  not  do  this. 

316. — Convert  a  part  of  the  creatin  into  creatinin  by  boiling 
fifteen  minutes  with  very  dilute  sulphuric  acid.  Neutralize  the 
acid  by  adding  powdered  barium,  carbonate  as  long  as  it  effer- 
vesces. Evaporate  to  dryness  on  a  water-bath  and  extract  the 
creatinin  from  the  residue  with  strong  alcohol.  Upon  evaporat- 
ing, the  creatinin  is  left  in  the  form  of  crystals. 

317. — Dissolve  a  little  of  the  creatinin  in  a  small  amount  of 
water,  add  a  solution  of  zinc  chlorid,  and  allow  to  stand.  Charac- 
teristic crystals  in  clusters  or  rosettes  appear.  They  are  a  double 
salt  of  creatinin  and  zinc  chlorid. 

318. — To  a  creatinin  solution  add  a  few  drops  of  a  freshly- 
prepared  solution  of  sodium  nitroprussid,  then,  drop  by  drop, 
dilute  sodium  hydrate.  The  liquid  becomes  ruby  red,  soon  chang- 
ing to  straw  color.  If  it  is  now  strongly  acidified  with  acetic  acid 
and  boiled,  it  becomes  green,  then  blue.  This  is  WeyPs  test. 

319. — To  a  solution  of  creatinin  add  a  few  drops  of  sodium 
hydrate,  then  of  picric-acid  solution.  A  red  color  is  obtained. 
This  is  Jaffe's  test. 

320. — With  a  needle  tease  out  some  shreds  of  muscle 
from  a  recently  killed  frog.  Place  them  on  a  microscope 
slide  and  expose  for  a  few  minutes  to  ammonia  gas  from  a 
strong  solution  of  the  hydrate.  When  covered  with  a  cover- 
glass  and  examined  they  are  seen  to  contain  stellate  crys- 
stals  of  ammonium  magnesium  phosphate,,  NH4MgP04. 
Explain  its  production. 

THE  BEAIN. 

321. — Clean  the  brain  of  a  sheep,  pig  or  calf,  pulp  in  a  mortar, 
extract  several  hours  with  88-per-cent.  alcohol  and  filter.  The 
lecithin,  cholesterin  and  the  substance  known  as  protagon  pass 
into  the  filtrate.  Cool  this  to  — 5°,  when  they  solidify,  then  re- 
move them  by  filtration.  Dissolve  the  lecithin  and  cholesterin  in 
cold  ether  (danger  from  fire!).  The  so-called  protagon  remains. 
After  this  has  been  dried  fuse  a  portion  with  potassium  nitrate 


142  MILK. 

in  a  nickel  dish  or  crucible.  Dissolve  the  residue  in  water  and  test 
for  phosphoric  acid  with  magnesia  mixture;  or  test  for  the  same 
substance  after  acidifying  by  nitric  acid  with  ammonium  molyb- 
date.  Mix  another  portion  of  the  dry  protagon  in  a  glass  tube 
with  soda-lime,  hold  horizontally  by  a  clamp  and  heat.  Show  that 
it  contains  nitrogen  from  the  evolution  of  ammonia  as  is  demon- 
strated by  the  action  of  the  gas  on  red  litmus  paper. 

322. — Pulp  another  brain  and  warm  it  with  barium  hydrate 
solution  (saponification).  Filter.  The  mass  on  the  filter  contains 
cerebrin,  cholesterin,  barium  soap,  and  connective  tissue.  Heat 
this  with  alcohol  and  filter  while  hot,  thus  dissolving  out  the 
cholesterin  and  cerebrin.  When  the  liquid  cools  they  separate  and 
are  recognizable  by  their  crystalline  forms.  The  cholesterin  is  in 
flat  thin  plates,  the  cerebrin  in  clusters  of  needle-shaped  crystals. 

Extract  the  cholesterin  by  ether,  and  recrystallize.  Test  a 
part  of  the  cerebrin  by  heating  with  soda-lime  for  nitrogen  as  in 
Experiment  72.  Boil  a  part  for  an  hour  with  dilute  sulphuric  acid 
to  hydrolize  it;  then  show  its  reducing  power  with  Trommer's 
reagent.  Observe  that  before  hydrolysis  it  does  not  reduce. 


MILK. 

The  solids  of  milk  are  partly  dissolved  and  partly  in 
suspension  in  the  liquid.  Of  the  dissolved  -constituents  the 
most  important  are  milk-sugar,  an  albumin,  a  globulin,  and 
some  mineral  salts.  Among  the  suspended  compounds 
are  casein,  fat,  and  calcium  phosphate.  The  average 
amount  of  solids  in  normal  cows'  or  human  milk  is  12  or 
13  per  cent,  by  weight.  The  reaction  of  fresh  cows'  or 
human  milk  is  nearly  neutral,  or  may  be  amphoteric  to 
litmus;  that  is,  it  turns  red  paper  blue  and  blue  paper  red. 

The  specific  gravity  should  be  between  1.029  and 
1.033  at  15°,  and  of  milk  which  has  been  skimmed  after 
standing  twenty-four  hours  it  should  be  between  1.0325 
and  1.0365.  Thus  the  removal  of  fat  raises  the  specific 


MILK.  143 

gravity  and  the  addition  of  water  lowers  it.  The  average 
percentage  composition  of  milk  is  given  by  Konig  as  fol- 
lows : — 

Cows', — water,  87.17;  proteins,  3.55;  fats,  3.69;  lac- 
tose, 4.88;  mineral  matter,  0.71. 

Human,— water,  87.41;  proteins,  2.29;  fats,  3.78;  lac- 
tose, 6.21;  mineral  matter,  0.31. 

Casein,  which  is  a  nucleoalbumin,  is  not  in  true  solu- 
tion in  milk,  since  it  can  be  filtered  out  by  unglazed  por- 
celain, though  not  by  filter-paper.  It  is  precipitated  by 
weak  acids,  as  is  seen  when  the  milk  becomes  sour,  but  is 
not  coagulated  by  boiling.  Eennin  breaks  up  the  casein 
into  two  compounds:  an  albumose  and  an  insoluble  cal- 
cium compound  (paracasein  calcium,  or  cheese).  Coagu- 
lated human  casein  is  not  as  hard  as  that  of  cows.  The 
difference  is  partly  due  to  its  chemical  composition,  but 
largely  to  the  fact  that  cows'  milk  contains  more  casein 
and  calcium  than  human  milk.  It  can  be  made  to  form 
a  soft  and  spongy  coagulum  similar  to  the  human  by  dilu- 
tion or  by  the  removal  of  the  calcium  compounds. 

The  fats  of  milk  are  a  mixture  of  stearin,  palmitin, 
and  olein,  with  a  small  amount  of  the  glycerids  of  some 
lower  members  of  the  fatty-acid  series, — butyric,  caproic, 
caprylic,  capric,  etc.  The  fat  exists  as  an  emulsion,  a  coat- 
ing of  albumin  keeping  the  globules  separate.  They  may 
be  made  to  collect  by  dissolving  this  coating  by  a  chemical 
agent,  like  sulphuric  acid.  Babcock's  method  for  deter- 
mining the  percentage  of  fat  in  milk  is  based  upon  this 
principle.  Here  the  volume  of  the  fat  is  measured  and 
this  gives  the  relative  amount  in  the  milk.  If  the  fat  rises 
for  twenty-four  hours  without  such  decomposition  it  should 
form  a  layer  10  or  15  per  cent,  of  the  depth  of  the  milk,  if 
the  latter  is  normal. 


144  MILK. 

The  method  of  obtaining  the  milk-sugar  has  been 
given.  (Experiment  34.) 

323. — Test  the  reaction  of  fresh  milk  to  red  and  blue 
litmus-paper. 

324. — Determine  the  specific  gravity  with  an  accurate 
urinometer. 

325. — Eemove  the  fat  from  milk  by  a  centrifuge,  or 
after  standing,  and  determine  the  specific  gravity  again. 

326. — Try  the  same  test  after  adding  from  10  to  25 
per  cent,  of  water  to  the  milk. 

327. — In  a  weighed  porcelain  or  platinum  crucible  evaporate 
10  cubic  centimeters  of  milk  to  dryness  on  a  water-bath  and  weigh 
quickly  to  find  the  amount  of  the  total  solid  matter.  The  drying 
will  take  place  much  more  rapidly  if  a  weighed  quantity  (about  20 
grammes)  of  dried  sand  is  added,  but  the  residue  cannot  be  used 
for  the  next  experiment. 

328. — Heat  the  dried  substance  in  the  crucible,  at  first  gently, 
then  until  no  black  remains.  The  residue  is  the  mineral  matter, 
or  ash.  There  should  not  be  over  1  per  cent,  of  the  weight  of  the 
milk. 

329. — Compare  the  action  of  rennin  upon  cows'  and 
human  milk.  Try  the  rennin  in  cows'  milk  to  which  50 
per  cent,  of  water  and  a  few  drops  of  ammonium  oxalate 
have  been  added  to  remove  the  calcium  salts. 

330. — To  separate  the  nitrogenous  constituents  of 
milk  first  precipitate  the  casein  by  saturating  the  milk 
(skimmed  milk  can  be  used)  with  sodium  chlorid.  Filter 
and  to  filtrate  add  powdered  magnesium  sulphate  as  long  as 
it  dissolves,  stirring  meanwhile.  This  precipitate  is  para- 
globulin,  the  same  compound  that  is  found  in  the  blood. 
Filter  and  apply  the  globulin  tests  (Experiments  98  to 
101).  Acidify  the  filtrate  with  a  few  drops  of  dilute  acetic 
acid  and  boil.  The  albumin  of  milk — lactalbumin — is  co- 


MILK.  145 

agulated.  To  determine  the  quantity  of  protein  subtract 
the  sum  of  fats,  sugar,  and  ash  from  the  total  solids. 

331. — Examine  with  the  microscope  a  drop  of  milk 
under  a  cover-glass. 

332. — Destroy  the  emulsion  by  adding  10  cubic  centi- 
meters of  concentrated  sulphuric  acid  to  an  equal  volume 
of  milk.  Let  it  stand,  and  the  fat  rises  to  the  top  in  large 
globules.  The  separation  is  complete  in  a  few  minutes  if 
a  centrifuge  is  used.  The  volume  of  the  fat  can  be  better 
seen  by  using  a  narrow-necked  flask  and,  after  mixing  with 
the  acid,  nearly  filling  with  warm  water. 

333. — Fill  a  100-cubic-centimeter  graduated  cylinder 
to  the  upper  mark  with  milk  and  let  it  stand  twenty-four 
hours.  There  should  be  10  or  15  cubic  centimeters  of 
cream. 

334.  To  DETERMINE  THE  PERCENTAGE  OF  LACTOSE 
IN  MILK. — Dilute  20  cubic  centimeters  of  milk  to  400 
cubic  centimeters  with  water.     Drop  in  acetic  acid  slowly 
until  it  coagulates;   then  pass  carbon  dioxid  gas  into  the 
liquid  fifteen  minutes  and  let  it  stand  until  it  settles  clear. 
Filter  and  wash;    coagulate  the  albumin  and  globulin  in 
the  filtrate  by  boiling.    Filter,  wash,  and  use  the  filtrate  or 
a  part  of  it  for  the  sugar-determination  by  Fehling's  solu- 
tion as  in  the  determination  of  glucose  (Experiment  33). 
For  every  10  cubic  centimeters  of  the  solution  which  is 
decolorized  0.067  gramme  of  lactose  is  present. 

335.  BABCOCK'S  METHOD  FOB  THE  DETERMINATION  OF  FAT 
IN  MILK  OK  CREAM.— When  sufficient  milk  is  available,  17.6  cubic 
centimeters  may  be  taken  for  each  determination.     The  bottles  in 
which  it  is  treated  have  a  special  scale  etched  upon  the  rather  long 
neck. 

Mix  the  milk  thoroughly  by  pouring  it  several  times  from 
one  vessel  to  another.  With  a  pipette  graduated  at  17.6  cubic 


146  THE  URINE. 

centimeters  measure  the  milk  and  pour  it  into  the  graduated  flask, 
adding  as  much  as  90-per-cent.  sulphuric  acid  (sp.  gr.,  1.82).  Mix 
by  gently  shaking  until  the  curd  has  completely  dissolved,  then 
revolve  in  the  centrifuge  at  600  to  800  revolutions  per  minute  for 
six  or  seven  minutes.  Always  make  duplicate  tests,  placing  the 
two  bottles  opposite  each  other  in  the  machine.  Now  carefully 
fill  the  bottles  about  to  the  highest  graduation  with  hot  water, 
which  should  have  been  previously  made  ready,  and  whirl  again 
for  one  or  two  minutes.  Holding  the  bottle  in  a  perpendicular 
position,  read  on  the  scale  the  differences  between  the  upper  and 
lower  margins  of  the  fat  which  gives  the  percentage  present  in  the 
milk.  If  the  test  is  successful  the  fat  layer  is  clear  or  but 
slightly  cloudy. 

With  breast-milk  where  the  available  amount  is  frequently 
limited  smaller  flasks  and  less  milk  may  be  employed.  Cream 
must  be  diluted  5  to  10  times  with  water. 


THE  UEINE. 

The  urine  is  a  solution  which  contains  the  final  prod- 
ucts from  the  chemical  changes  in  progress  in  the  animal 
body.  A  part  of  these  are  excreted  in  the  expired  air  and 
from  the  skin,  and  a  still  smaller  part  through  the  mucous 
membrane  of  the  intestine,  but,  if  we  omit  the  carbon  di- 
oxid  from  the  lungs,  by  far  the  greater  proportion  of  these 
final  products  is  found  in  the  urine.  A  study  of  its  com- 
position and  variation,  therefore,  is  often  of  great  value 
in  judging  of  changes  which  are  going  on  in  the  body. 

Among  the  most  common  inorganic  constituents 
normally  found  are  the  chlorids,  sulphates,  and  phosphates 
of  sodium,  potassium,  calcium,  and  magnesium.  Of  the 
normal  organic  compounds  there  are  urea,  uric  acid  and 
its  salts,  creatinin,  etc.  The  following,  when  found  in 
more  than  minute  amounts,  may  be  regarded  as  patho- 
logical: Glucose,  albuminous  substances,  blood,  bile,  pus, 
fat,  mucin,  leucin,  and  tyrosin.  Others  which  are  more 


THE   URINE.  147 

rare  will  be  spoken  of  later.  All  of  these  either  are  taken 
as  such  into  the  body  with  the  food  or  are  formed  in  the 
body  by  chemical  action.  The  significance  of  each  may 
depend  upon  the  amount  which  is  present,  as  well  as  upon 
its  mere  presence  or  absence.  In  interpreting  the  mean- 
ing of  each  of  the  constituents  of  the  urine  its  method  of 
formation  must  be  considered,  as  well  as  the  factors  which 
may  cause  this  to  vary. 

Considerable  variations  are  found  in  the  composition 
of  urine  which  has  been  collected  at  different  times  of  the 
day.  That  which  is  passed  immediately  after  rising  may 
differ  from  that  excreted  an  hour  or  two  after  the  first 
meal  both  in  the  kind  and  amount  of  the  dissolved  solids. 
Sugar  and  albumin  are  more  commonly  excreted  after  a 
meal,  and  may  be  found  then,  yet  not  be  present  in  the 
night's  urine.  In  order  to  obtain  a  fair  sample  for  testing, 
the  urine  should  be  collected  for  twenty-four  hours  and, 
after  mixing,  a  part  taken  for  analysis.  In  all  quantitative 
determinations  the  volume  for  twenty-four  hours  must  be 
measured,  and  when  it  has  been  determined  how  much  of 
the  substance  is  present  in  the  portion  tested,  the  amount 
contained  in  the  whole  day's  urine  should  be  calculated. 
A  statement  of  the  percentage  alone  has  little  value  if  the 
quantity  of  the  urine  is  not  taken  into  account.  To  avoid 
fermentation  the  vessels  should  be  clean  and  the  tests 
should  be  made  as  soon  as  possible. 

The  average  volume  of  the  urine  in  twenty-four  hours 
is,  for  an  adult,  between  900  cubic  centimeters  and  1200 
cubic  centimeters  (30  and  40  ounces).  This,  however,  is 
subject  to  great  variations.  It  is  increased  by  diuretics,  by 
diseases,  like  diabetes  and  others;  it  is  diminished  in  febrile 
diseases,  in  acute  nephritis,  in  some  other  diseases  of  the 
kidneys,  and  usually  before  the  fatal  termination  of  a  dis- 


148  THE   URINE. 

ease.  Its  variation  gives  indications  of  the  progress  of  the 
disease.  The  volume  will  be  also  affected  by  the  amount 
of  drink  or  liquid  food  and,  in  general,  varies  inversely 
with  the  perspiration. 

From  the  presence  of  ferments,  the  urine  begins  to 
undergo  a  change  after  it  has  stood  a  few  hours.  The  re- 
action becomes  alkaline,  owing  to  the  production  of  am- 
monium carbonate  from  the  urea,  and  this  precipitates 
some  of  the  solids,  so  that  the  liquid  loses  its  transparency. 
This  and  other  decompositions  produce  disagreeable  odors. 

The  odor  of  normal  urine  is  characteristic.  Certain 
foods  and  medicines  change  this;  e.g.,  oil  of  turpentine 
gives  an  odor  of  violets.  When  it  putrefies  the  odor  is 
ammoniacal  and  offensive.  In  cystitis  it  is  ammoniacal 
when  passed.  In  suppurative  diseases  the  odor  may  be 
putrid. 

Fresh,  normal  urine  is  clear,  but  after  standing  a 
short  time  a  cloud  of  mucus  appears.  Pathologically  it 
may  be  cloudy  with  matters  which  settle  as  a  sediment. 
They  will  be  discussed  under  that  subject. 

The  color  of  urine  is  normally  some  shade  of  yellow, 
varying  from  nearly  colorless  to  reddish  yellow.  The 
former  is  true  of  urines  containing  much  water,  and  the 
latter  where  the  urine  is  concentrated  and  of  high  specific 
gravity.  The  latter  is  constant  in  febrile  conditions  and 
their  severity  can  here  often  be  judged  from  the  color. 
Pathologically  the  urine  assumes  many  other  shades. 
Presence  of  blood  gives  a  red  or,  when  methaBmoglobin  is 
present,  a  brown.  Jaundice  gives  a  greenish  cast  or 
brownish  green;  melanotic  cancer,  almost  black;  typhus 
or  cholera,  sometimes  blue,  from  indigo  formed  by  decom- 
position. Some  medicinal  or  poisonous  substances  change 
the  color;  thus  senna  or  rhubarb  gives  a  reddish  or  brown- 


SPECIFIC   GRAVITY.  149 

ish  color,  which  changes  to  blood-red  on  adding  an  alkali. 
Santonin  gives  a  yellow;  carbolic  acid  and  salol  a  dark 
green  to  black;  antipyrin  and  quinine  often  darken  it. 

The  specific  gravity  of  urine  varies  with  the  amount 
of  water  and  dissolved  solids.  With  an  increase  of  the 
water  it  approaches  1.000,  and  becomes  greater  as  the 
solids  increase.  Hence  it  is  easy  to  ascertain  the  amount 
of  the  solids  which  are  present.  If  the  second  and  third 
decimal  figures  of  the  specific  gravity  are  multiplied  by 
2.33  it  will  give  very  nearly  the  weight  of  dissolved  sub- 
stances in  one  thousand  parts  of  urine  (grammes  per  liter). 
Thus,  urine  of  sp.  gr.  of  1.021  contains  about  49  grammes 
in  a  liter. 

The  specific  gravity  varies  under  normal  conditions 
from  1.002  to  1.030.  It  is  usually  between  1.015  and 
1.025.  If  sugar  is  not  present  the  variation  in  specific 
gravity  is  due  almost  entirely  to  that  of  the  urea.  Clin- 
ically the  specific  gravity  of  urine  is  determined  by  an 
hydrometer,  called  a  urinometer,  which  consists  of  a  spin- 
dle weighted  so  as  to  float  in  pure  water  at  the  line  marked 
1.000.  The  specific  gravity  is  indicated  by  the  figures  on 
the  spindle  at  the  surface  of  the  liquid.  Urinometers 
should  always  be  tested  in  pure  water  and  if  they  are  not 
correct  the  reading  in  the  urine  must  be  changed  to  corre- 
spond with  the  error.  Since  the  specific  gravity  varies 
with  the  temperature  some  standard  temperature  must  be 
adopted.  Most  instruments  are  graduated  at  60°  F.  (15.6° 
C.).  The  urine  must  be  brought  to  this  temperature  be- 
fore testing  or,  if  accuracy  is  desired,  the  reading  corrected 
by  adding  1  in  the  fourth  decimal  place  for  every  degree 
Fahrenheit  above  60°  or  subtracting  1  for  each  degree 
below  60°.  In  order  to  obtain  accurate  results  the  degrees 
should  not  be  too  close  together  on  the  spindle. 


150  THE   URINE. 

The  importance  of  a  knowledge  of  the  specific  gravity 
is  rather  to  detect  marked  changes  in  the  urine  from  a 
series  of  observations  than  to  be  able  to  infer  the  presence 
of  some  abnormal  constituent,  like  glucose,  which  would 
certainly  be  found  by  the  subsequent  tests.  Thus,  in 
nephritis  a  decrease  in  specific  gravity  without  change  in 
the  volume  indicates  that  the  urea  is  not  being  excreted 
and  that  uremia  may  be  feared. 

336. — Test  the  accuracy  of  the  urinometer  in  water, 
then  take  the  specific  gravity  of  urine.  The  cylinder  must 
be  wide  enough  for  the  urinometer  to  float  in  it  without 
touching.  Foam  on  the  liquid  should  be  removed  by  a 
piece  of  filter-paper. 

337. — Test  with  an  accurate  urinometer  the  difference 
in  specific  gravities  of  freshly-passed  urine  when  at  a  tem- 
perature of  from  95°  to  98°  F.  and  that  at  60°  F.  or  below. 

The  reaction  of  normal  mixed  human  urine  passed 
during  twenty-four  hours  is  acid.  Quantitative  determina- 
tions of  the  salts  in  the  urine  show  that  the  bases  (kations) 
are  not  present  in  large  enough  amounts  to  replace  all  the 
hydrogen  of  the  acids.  This  fact  is  commonly  expressed  in 
the  statement  that  the  acid  reaction  is  due  to  acid  salts, 
principally  acid  phosphates  of  sodium  and  potassium. 
However,  since  these  as  well  as  the  other  inorganic  com- 
pounds are  more  or  less  dissociated,  it  is  preferable  to  say 
that  the  hydrogen  ions  (hydrions)  cause  the  acidity.  The 
administration  of  alkaline  drugs  is  followed  by  the  urine's 
becoming  less  acid  or  even  alkaline.  The  same  effect  is 
produced  by  vegetable  foods.  These  contain  the  potas- 
sium salts  of  organic  acids — citric,  malic,  tartaric,  and 
others  which  are  oxidized  to  potassium  carbonate  in  the 
system.  A  similar  result  is  brought  about  a  short  time 
after  a  hearty  meal,  when  hydrochloric  acid  is  being  set 


REACTION.  151 

free  from  its  salts  in  the  mucous  cells  of  the  stomach. 
The  bases  which  are  freed  at  the  same  time  remain  to  in- 
crease the  alkalinity  of  the  blood.  Part  of  them  pass  into 
the  urine,  producing  the  "alkaline  tide,"  or  alkaline  reac- 
tion, which  is  often  noticed  at  this  time.  The  urine  of 
herbivorous  animals  is  normally  alkaline  from  this  cause. 
On  the  other  hand,  an  acid  food  or  one  from  which  acids 
are  produced  during  its  decomposition  in  the  body  will 
increase  the  acidity.  Such  a  one  is  lean  meat,  which  con- 
tains acid  potassium  phosphate,  and  also  sulphur  and  phos- 
phorus compounds,  which  form  sulphuric  and  phosphoric 
acids  by  oxidation.  Hence  the  reaction  of  the  urine  may 
be  to  a  considerable  extent  regulated  by  the  selection  of 
foods. 

Upon  standing  all  urine  becomes  alkaline  by  fermenta- 
tion. This  is  produced  by  the  action  of  a  number  of  micro- 
organisms upon  the  urea,  resulting  in  the  formation  of 
ammonium  carbonate: — 

CO(NH2)2  +  2H20  =  (NH4)2C03. 

If  these  ferments  are  introduced  into  the  bladder  by 
an  unclean  catheter  the  same  action  is  often  produced 
there.  In  chronic  inflammation  of  the  urinary  tract  am- 
monium carbonate  is  usually  present.  The  latter  alka- 
linity— from  ammonium  carbonate — can  be  distinguished 
from  that  produced  by  sodium  and  potassium  salts  by  the 
litmus-paper's  resuming  its  red  color  after  drying,  if  am- 
monia were  the  alkali,  but  not  otherwise. 

In  determining  the  degree  of  acidity  of  the  urine  by 
the  use  of  a  standard  alkaline  solution,  litmus  cannot  be 
used  to  indicate  when  the  neutralization  is  complete,  on 
account  of  the  interference  of  the  phosphates. 


152  THE   URINE. 

Excessive  acidity1  of  the  urine  causes,  in  time,  an  irri- 
tation of  the  urinary  passages,  and  is  favorable  to  the 
formation  of  uric  acid  concretions.  Continued  alkalinity 
makes  a  sediment  in  the  urine,  and  tends  to  produce  phos- 
phatic  calculi.  It  also  produces  irritation  or  inflammation 
of  the  mucous  membrane. 

338. — Test  the  reaction  of  urine  with  sensitive  litmus- 
paper,  and  if  alkaline  determine  whether  it  is  caused  by 
ammonium  carbonate  by  the  paper's  turning  red  again 
after  drying,  or  whether  a  sodium  or  potassium  compound 
is  the  alkali  by  the  paper's  remaining  blue  on  drying. 

339.  To  DETERMINE  THE  ACIDITY  OF  URINE. — To  50  cubic 
centimeters  in  a  flask  add  25  cubic  centimeters  of  l/10  normal 
sodium  hydrate,  and  heat  to  boiling,  then  remove  the  flame. 
Thereupon  add  25  cubic  centimeters  of  barium  chlorid  solution 
of  about  5  or  10  per  cent.  Filter  through  a  dry  filter,  and  take 
50  cubic  centimeters  of  the  filtrate,  corresponding  to  25  cubic 
centimeters  of  the  urine,  for  testing.  Dilute  it  to  about  250  cubic 
centimeters  with  water,  add  a  few  drops  of  phenolphthalein  for 
an  indicator,  then  from  a  burette,  Vio  normal  sulphuric  acid  until 
the  red  color  is  just  destroyed.  Subtracting  the  number  of  cubic 
centimeters  of  acid  used  from  12.5,  the  number  of  cubic  centimeters 
of  standard  sodium  hydrate  in  the  half  of  the  liquid  used,  gives 
the  number  of  cubic  centimeters  of  sodium  hydrate  neutralized  by 
the  acid  in  25  cubic  centimeters  of  urine. 

340. — Collect  the  urine  of  the  day  in  periods  of  three  hours 
each  and  determine  the  variations  in  its  acidity. 

341.  —  Take  internally  sodium  acetate  in  2  to  3 
gramme  doses,  and  note  its  effect  upon  the  reaction  of 
the  urine. 

UKEA. 

About  86  per  cent,  of  the  nitrogen  in  the  urine  of  a 
healthy  man  has  been  found  to  be  in  the  urea,  CO(NH2).,. 
Under  pathological  conditions,  however,  it  may  vary  greatly 


UREA.  153 

from  this.  The  absolute  weight  varies  between  20  and  40 
grammes  daily,  being  somewhat  less  for  a  woman  than  for 
a  man.  In  round  numbers,  we  can  say  that  it  is  about  one 
ounce  in  twenty-four  hours  for  the  adult  male. 

Urea  crystallizes  in  long,  colorless,  rhombic  prisms. 
It  is  easily  soluble  in  alcohol  and  in  water;  hence  it  never 
forms  a  sediment.  It  forms  double  compounds  with  acids, 
some  .of  which,  like  the.  nitric  and  oxalic  acid  compounds 
are  not  easily  soluble,  and  are  used  in  separating  the  urea 
from  urine.  It  forms  similar  insoluble  compounds  with 
many  salts  of  the  heavy  metals,  mercury,  copper,  etc. 

When  urea  is  brought  into  contact  with  a  hypobro- 
mite  or  a  hypochlorite,  it  is  decomposed  into  carbon  dioxid, 
nitrogen,  and  water:  — 


CO(NH)2  +  3NaOBr  =  3NaBr  +  C02  +  N2  +  2H20. 

This  decomposition  is  made  use  of  to  determine  the 
amount  of  urea  in  urine  by  measuring  the  volume  of  the 
nitrogen  set  free.  There  are  a  great  number  of  modifica- 
tions in  form  of  the  apparatus  employed,  —  Hiifner's, 
Doremus's,  Squibb's,  and  many  others,  —  all  based  upon  the 
same  principle.  They  do  not  give  absolutely  accurate  re- 
sults, but  are  sufficiently  exact  for  clinical  tests,  and  have 
the  advantage  of  requiring  but  a  short  time  for  their  exe- 
cution. Where  it  is  desirable  to  learn  accurately  the 
amount  of  nitrogenous  compounds  excreted,  it  is  best  to 
find  the  total  nitrogen  by  KjeldahPs  method.  The  solu- 
tion of  sodium  hypobromite  should  be  freshly  prepared 
from  bromin  and  sodium  hydrate,  as  it  decomposes  on 
standing. 

Doremus's  ureometer  for  determining  the  percentage 
of  urea  in  urine  consists  of  a  short  graduated  tube  closed 
at  the  upper  end.  Below  it  is  bent  upward  and  expands 


154 


THE   URINE. 


to  a  bulb.  The  graduations  represent  for  each  division 
0.001  gramme  of  urea.  That  is,  0.001  gramme  of  urea 
evolves  enough  nitrogen  to  fill  one  division.  Since  one 
cubic  centimeter  of  urine  is  used,  weighing  very  nearly  a 
gramme,  nitrogen  to  fill  one  division  corresponds  very 
nearly  to  0.1  per  cent,  of  urea  in  the  urine.  With  the  tube 
is  furnished  a  1-cubic-centimeter  dropping  pipette. 


D 


Apparatus  for  Determining  Urea  in  Urine. 

An  apparatus  can  be  simply  and  cheaply  made,  after 
the  principle  of  SquibFs,  from  two  4-ounce  wide-mouth 
bottles  (see  figure).  One  of  these  (A)  contains  a  vial  (C), 
which  serves  to  hold  the  urine. 

Outside  (7,  in  A,  is  placed  the  solution  of  sodium  hy- 
pobromite.  B  contains  water  and  is  connected  with  A  by 
a  rubber  tube.  When  the  rubber  stoppers  are  tightly  in- 
serted the  urine  is  brought  into  contact  with  the  hypo- 
bromite  by  tipping  A,  the  nitrogen  of  the  urea  being 
liberated.  This  forces  from  B  an  equal  volume  of  water. 
The  water  is  collected  from  the  tube  D,  and  when  meas- 


SOURCE   OF   UREA.  155 

ured  gives  the  volume  of  nitrogen  set  free.  The  glass 
tubes  (D,  etc.)  should  be  of  small  diameter.  If  there  is 
no  leak  in  the  apparatus,  pressing  the  stopper  into  the 
bottle  A  will  force  water  from  B  into  the  tube  D,  and  it 
should  remain  full,  without  running  out,  as  long  as  the 
apparatus  is  not  disturbed. 

Urea  is  probably  formed  in  the  liver.  Its  source  is 
the  nitrogenous  compounds  of  the  food  and  the  tissues, 
including  the  blood,  most  of  the  nitrogen  of  such  com- 
pounds being  excreted  from  the  body  in  the  urea.  Hence 
any  increase  in  the  destruction  of  these  substances  is  ac- 
companied by  an  increased  formation  of  urea  and  vice 
versa.  For  this  reason  the  urea  is  considered  as  a  measure 
of  the  decomposition  of  the  proteins  in  the  body. 

Some  things  which  bring  about  an  increased  decom- 
position of  proteins  are:  a  large  amount  of  nitrogenous 
food,  like  meat;  excessive  exercise,  which  causes  a  de- 
struction of  tissue,  though  here  the  urea  is  not  propor- 
tional to  the  exertion;  fevers  and  inflammations  up  to  the 
crisis,  owing  to  the  rapid  loss  of  muscular  tissue.  After 
the  crisis  it  is  diminished.  In  phosphorus  poisoning  and 
diabetes  mellitus  the  urea  is  excessive  for  the  same  reason. 
A  greater  excretion  of  water,  either  from  excessive  drink- 
ing or  diuretics,  carries  with  it  a  larger  amount  of  urea, 
which  seems  to  be  thus  washed  out  of  the  system. 

On  the  .other  hand,  less  urea  is  excreted  during  hunger 
and  sleep,  when  the  metabolism  of  the  body  is  lessened. 
Interference  with  the  excretory  power  of  the  kidneys  like- 
wise diminishes  the  urea.  This  is  seen  in  acute  nephritis 
and  other  diseases  of  the  kidneys.  In  such  cases  the  pro- 
duction of  urea  is  not  stopped,  but  it  accumulates  in  the 
system,  often  being  accompanied  by  urasmic  poisoning. 
Since  the  urea  is  formed,  at  least  in  part,  in  the  liver,  we 


156  THE    URINE. 

find  that  less  is  excreted  in  carcinoma  and  cirrhosis  of  this 
organ. 

The  fermentation  of  urea  to  ammonium  carbonate, 
caused  by  the  action  of  micro-organisms,  has  been  already 
referred  to. 

PREPARATION   OF   UREA. 

342.  PROM  URINE. — (a)  If  only  a  small  amount  is 
desired,  evaporate  half  a  test-tubeful  of  urine  to  dryness 
on  the  water-bath.  Dissolve  the  urea  from  the  residue 
with  95-per-cent.  alcohol.  Allow  a  drop  of  the  alcoholic 
filtrate  to  evaporate  on  a  microscope-slide  without  the  aid 
of  heat.  Examine  the  crystals  under  the  microscope.  If 
the  form  is  not  distinct,  dissolve  in  a  drop  of  water  and 
again  observe  the  crystals  after  this  has  evaporated.  Add 
a  drop  of  dilute  nitric  acid  to  the  slide,  let  it  stand  a  few 
minutes,  then  examine  the  crystals  of  urea  nitrate.  (Plate 
1,6.) 

(b)  A  larger  quantity  can  best  be  obtained  by  evapo- 
rating half  a  liter  to  a  liter  of  urine  to  a  thin  syrup  upon 
the  water-bath,  then  cooling  it  in  ice-water,  and  adding 
about  three  times  its  volume  of  nitric  acid  of  a  specific 
gravity  of  1.3  which  has  been  boiled  to  expel  the  oxids 
of  nitrogen  and  cooled  with  ice-water.  Filter  off  the 
urea  nitrate  through  an  asbestos  or  glass-wool  filter,  wash- 
ing with  a  small  quantity  of  ice-cold  concentrated  nitric 
acid.  Dissolve  the  crystals  in  hot  water  and  decolorize  by 
chlorin-water  or  a  small  quantity  of  potassium  chlorate. 
Add,  then,  small  portions  of  pure  barium  carbonate  as  long 
as  it  dissolves  and  until  the  liquid  is  neutral.  Evaporate 
the  whole  upon  the  water-bath  to  dryness.  Pulverize  the 
residue  and  dissolve  the  urea  in  absolute  alcohol,  which 
does  not  dissolve  the  barium  nitrate.  If  the  alcoholic  solu- 


SYNTHESIS   OF   UREA.  157 

tion  is  colored  it  can  be  decolorized  by  filtering  it  through 
bone-black.  Distill  off  the  alcohol  or  allow  it  to  evaporate 
to  obtain  the  urea. 

343.  SYNTHETICALLY. — Coarsely  powder  50  grammes 
of  potassium  ferrocyanid  and  heat  in  an  iron  dish  over  a 
Bunsen  flame,  stirring  continually,  until  it  has  become  a 
white  powder  and  the  lumps  show  no  yellow  color  when 
they  are  broken.  If  it  turns  brown  the  heat  is  too  high. 
Pulverize  the  mass  as  finely  as  possible  in  a  mortar.,  mix  it 
thoroughly  with  half  its  weight  of  finely-powdered  man- 
ganese dioxid,  and  heat  in  an  iron  dish  under  the  hood, 
stirring  meanwhile,  until  the  mass  glows  and  becomes  thick 
and  sticky.  Heat  until  a  small  test  dissolved  in  hydro- 
chloric acid  gives  no  blue  color  with  ferric  chlorid.  Then 
allow  it  to  cool ;  dissolve  the  potassium  cyanate,  which  has 
thus  been  formed  with  cold  water.  Convert  this  into  am- 
monium cyanate  by  the  addition  of  38  grammes  of  dry 
ammonium  sulphate.  Filter  and  evaporate  upon  the  water- 
bath  at  about  60°  to  70°,  at  which  temperature  ammonium 
cyanate  is  converted  into  urea.  The  potassium  sulphate 
crystallizes  out  first,  and  should  be  removed  from  time  to 
time.  At  last  evaporate  to  dryness  and  dissolve  out  the 
urea  with  absolute  alcohol  as  before. 

344. — Mix  a  few  of  the .  dry  crystals  with  soda-lime 
and  heat  in  a  dry  test-tube.  The  presence  of  nitrogen  is 
shown  by  the  evolution  of  ammonia. 

345. — Warm  some  crystals  of  pure  urea  in  a  dry  test- 
tube.  It  melts,  then  decomposes,  yielding  ammonia,  which 
can  be  identified  by  litmus-paper.  When  the  substance  has 
solidified,  cool  it,  dissolve  in  water,  make  alkaline  with 
sodium  hydrate,  and  add  a  few  drops  of  copper  sulphate 
solution.  The  color  is  due  to  the  presence  of  biuret,  IS[II2- 
CONHCONH2.  Write  the  equation  for  its  formation. 


158  THE   URINE. 

346. — To  5  cubic  centimeters  NaOH  add  a  drop  of 
bromin,  and  after  this  has  dissolved  a  few  crystals  of  urea. 
Explain  the  result. 

347.  PREPARATION  OF  SODIUM  HYPOBROMITE. — In  a 
thin  glass  flask  or  beaker  containing  20  cubic  centimeters 
of  water  dissolve  8  grammes  of  sodium  hydrate.  Cool, 
and  from  a  dropping  pipette  or  funnel  add  slowly  2  cubic 
centimeters  of  bromin,  stirring  or  shaking  meanwhile. 
Handle  the  bromin  under  a  hood  or  in  a  draft  of  air  to 
avoid  the  vapors,  which  are  especially  irritating  to  the 
eyes  and  lungs.  As  the  bromin-gas  is  heavy,  it  should  be 
held  below  the  level  of  the  face  while  pouring,  rather  than 
above. 

348. — Determine  the  percentage  of  urea  in  urine  by 
the  use  of  Doremus's  ureometer.  First  fill  the  tube  with 
the  hypobromite  solution  and  invert  it,  having  no  more 
of  the  liquid  in  the  bulb  than  is  necessary  to  keep  the  tube 
full.  Fill  the  pipette  exactly  to  the  mark  with  urine,  in- 
sert the  lower  end  into  the  ureometer,  and  slowly  and 
steadily  force  the  urine  out  by  compressing  the  rubber 
bulb.  The  urine,  being  the  lighter  liquid,  rises  in  the  ureo- 
meter and  the  urea  is  immediately  decomposed.  The  car- 
bon dioxid  is  dissolved  in  the  solution  and  only  the  nitro- 
gen is  collected.  No  gas-bubbles  should  be  allowed  to 
escape  into  the  ureometer-bulb  or  back  into  the  pipette, 
thereby  causing  a  loss.  When  the  foam  has  disappeared, 
read  off  the  quantity  of  gas  and  calculate  the  percentage 
of  urea.  Duplicate  tests  should  not  differ  more  than  0.1 
per  cent.  If  the  volume  of  urine  in  twenty-four  hours  is 
known,  calculate  the  weight  of  urea  excreted  in  that  time. 

349. — Determine  the  amount  of  urea  in  urine  by  ap- 
paratus, page  154.  Fill  B  nearly  full  of  water.  Into  C 
by  a  pipette  put  exactly  2  cubic  centimeters  of  urine.  Out- 


DETERMINATION  OF   UREA  AND   NITROGEN.  159 

side  of  C  in  A  put  20  or  25  cubic  centimeters  of  the  hypo- 
bromite.  Insert  the  stopper  (E)  tightly,  thus  filling  the 
tube  D  with  water.  If  it  remains  full,  showing  that  the 
apparatus  is  tight,  place  an  empty  beaker  under  D  and 
gently  mix  the  urine  and  hypobromite.  Avoid  as  much 
as  possible  raising  the  temperature  by  holding  the  bottle 
in  the  hand,  as  the  expansion  of  the  gas  causes  a  consid- 
erable error.  Allow  it  to  stand  until  no  more  water  passes 
from  D,  which  must  remain  full  of  water  during  the  whole 
test;  then  measure  the  expelled  water  in  a  graduated  cy- 
linder. 

One  gramme  of  urea  contains  371  cubic  centimeters 
of  nitrogen;  but,  when  it  is  decomposed  in  this  manner, 
only  about  354  cubic  centimeters  are  obtained.  For  ordi- 
nary clinical  purposes  the  percentage  of  urea  in  urine  can 
be  calculated  from  the  following  formula:  — 

100  x  number  of  c.  c.  of  N 
percentage  of  urea  =  " 


350.    DETERMINATION  OF  THE  TOTAL  NITROGEN  IN  URINE 
(KJELDAHL'S  METHOD*1).  —  I.  Prepare  the  following  solutions:  — 

1.  Standard  sulphuric  acid  containing  about  25  grammes  per 
liter,  of  which  the  strength  has  been  accurately  determined. 

2.  Standard  ammonia,  of  which  about  five  volumes  are  neces- 
sary to  neutralize  one  of  the  acid.    Determine  this  accurately  and 
calculate  the  amount  of  ammonia  by  weight  in  1  cubic  centimeter. 

3.  Sodium  hydrate  free  from  ammonia,  and  nitric  acid,  about 
270  grammes  per  liter. 

4.  Congo-red,  of  which  the  solution  contains  0.2  gramme  in 
100  cubic  centimeters.  This  is  turned  red  by  alkalies  and  blue  by 
acids  —  the  opposite  of  litmus. 


1  This  method  can  be  used  for  finding  the  amount  of  N  in 
most  animal  and  vegetable  compounds. 


160  THE    URINE. 

Have  also  at  hand: — 

1.  Sulphuric  acid,  sp.  gr.  1.84,  free  from  compounds  of  nitro- 
gen. 

2.  Yellow  mercuric  oxid. 

3.  Powdered  potassium  permanganate. 

4.  Crystallized  sodium  thio-sulphate. 

II.  Operation. — With  a  pipette  measure  accurately  5  cubic 
centimeters  of  urine.  Place  it  in  a  flask  holding  about  250  cubic 
centimeters,  best  of  hard  (Bohemian)  glass.  Add  0.4  gramme  of 
mercuric  oxid  and  10  cubic  centimeters  of  the  concentrated  sul- 
phuric acid.  Lay  the  flask  in  a  slanting  position  on  a  wire  gauze 
over  a  flame  small  enough  to  just  bring  it  to  boiling.  Perform  this 
operation  under  a  hood  or  where  there  is  a  good  draft  to  carry  away 
the  fumes  of  the  acid.  Continue  the  heating  until  the  liquid  is 
colorless  or  straw-colored,  which  may  require  from  thirty  minutes 
to  an  hour.  Then  remove  the  flask  from  the  flame  and  very 
slowly  add  to  it  a  small  amount  of  the  powdered  permanganate 
until  it  is  colored  reddish  or  greenish.  Allow  it  to  cool  and  pour 
it  into  an  800-cubic-centimeter  flask  which  contains  from  200  to 
300  cubic  centimeters  of  distilled  water,  rinsing  the  small  flask 
into  the  large  one.  The  organic  matter  has  been  oxidized  in  this 
process,  the  nitrogen  being  converted  into  ammonia,  which  is  con- 
tained in  solution  as  ammonium  sulphate. 

The  ammonia  is  now  to  be  set  free  by  sodium  hydrate,  then 
distilled  into  a  known  amount  of  standard  acid,  and  its  amount 
found  by  ascertaining  the  loss  of  strength  of  the  acid  through  its 
neutralization  by  the  ammonia.  For  this  purpose  a  Liebig  con- 
denser is  to  be  arranged  so  that  the  flask  can  be  connected  with 
the  upper  end  by  means  of  a  bent  tube;  this  should  be  at  least 
Y4  inch  in  diameter  inside  and  a  foot  long,  to  prevent  small  drops 
of  the  boiling  liquid's  being  carried  over.  The  insertion  of  a  bulb 
between  the  flask  and  condenser,  also  having  the  lower  end  of  the 
bent  tube,  in  the  flask,  cut  off  obliquely,  will  aid  in  preventing  this. 
Add,  now,  to  the  liquid  in  the  flask  a  few  fragments  of  granulated 
zinc  to  make  it  boil  more  quietly,  about  a  gramme  of  sodium  thio- 
sulphate  to  precipitate  the  mercury,  and  80  cubic  centimeters  of 
the  sodium  hydrate,  or  enough  to  make  it  alkaline.  Connect 
immediately  with  the  condenser  through  which  a  stream  of  cold 
water  is  flowing  and  distill  into  a  400  or  500  cubic  centimeter 


URIC   ACID.  161 

conical  flask  (Erlenmeyer)  which  contains  exactly  10  cubic  centi- 
meters of  the  standard  acid  (not  the  concentrated!).  Continue 
the  distillation  until  at  least  half  has  been  distilled  over  and  the 
distillate  coming  from  the  condenser  no  longer  turns  red  litmus- 
paper  blue. 

Then  find  how  much  ammonia  has  been  taken  up  by  the 
standard  acid.  To  do  this  add  a  few  drops  of  Congo-red  solution  to 
the  distillate.  It  will  be  colored  red,  because  of  the  acid  reaction. 
From  a  burette  add  the  standard  ammonia,  stirring  meanwhile, 
until  the  red  just  changes  to  a  blue,  when  the  liquid  is  neutral. 
Subtract  the  number  of  cubic  centimeters  of  ammonia  used  from 
the  number  which  are  required  to  neutralize  10  cubic  centimeters 
of  the  standard  acid.  The  difference  represents  the  volume  of 
standard  ammonia  equal  to  that  which  was  distilled  from  the 
oxidized  urine.  Calculate  the  weight  of  NH3  in  this.  Fourteen- 
seventeenth  of  the  NH3  is  the  weight  of  the  nitrogen  in  5  cubic 
centimeters  of  urine.  Calculate  the  percentage. 

URIC  ACID  ("LITHIC  ACID"). 

Uric  acid  is  normally  present  in  solution  in  the  urine 
of  mammals.  With  birds  and  snakes  it  is  the  principal 
nitrogenous  excretory  product.  Its  formula  is  C5H4N403 
and  the  constitution  of  the  molecule  is  probably 


—  CO 


It  is  consequently  2-6-8-trioxypurin. 

The  daily  amount  varies  much,  but  averages  from  0.2 
to  0.8  gramme.  Except  that  it  must  be  formed  from  the 
nitrogenous  compounds  in  the  body,  we  know  little  of  its 
production  or  of  the  cause  and  significance  of  its  variations. 


162  THE   URINE. 

Uric  acid  is  comparatively  insoluble  in  water  or  acids, 
but  dissolves  readily  in  the  fixed  alkalies,  forming  salts  of 
uric  acid,  or  urates.  In  the  urine  the  acid  exists  in  the 
form  of  these  salts  or  united  with  some  organic  base.  It 
is  a  dibasic  acid  like  sulphuric  acid,  having  two  atoms  of 
hydrogen  which  can  be  replaced  by  metals.  It  can  thus 
have  two  series  of  salts,  the  acid  and  the  normal,  corre- 
sponding to  HKS04  and  K2S04.  Of  these  classes  the  nor- 
mal salts  are  quite  soluble  in  water,  but  the  acid  salts  do 
not  dissolve  so  easily.  The  acid  can  be  set  free  from  its 
salts  by  the  use  of  a  stronger  acid.  The  solubility  of  the 
acid  salts  is  much  less  in  cold  water  than  in  warm.  Con- 
sequently they  frequently  separate  from  urine  which  was 
clear  when  passed  but  has  stood  in  a  cold  room,  and  they 
can  then  be  redissolved  by  warming. 

When  it  is  pure,  uric  acid  exists  in  the  form  of  color- 
less crystals.  As  it  is  found  in  the  urine,  it,  as  well  as  its 
salts,  is  always  colored  yellow  to  brown  by  the  coloring 
matter  which  has  been  carried  down  from  the  urine.  The 
simplest  form  of  crystals  is  tabular  with  curved  sides  and 
pointed  ends.  These  are  frequently  united  at  right  angles, 
making  a  star-shaped  form,  two  of  the  rays  often  being 
smaller  than  the  other  two.  In  urinary  sediments  many 
crystals  may  be  united,  making  a  rosette-like  form.  In 
strongly-acid  urine  the  crystals  sometimes  have  jagged 
edges  like  the  teeth  of  a  broken  comb.  Many  different 
forms  may  be  obtained  by  precipitating  with  various 
strengths  of  acid.  (Plate  II,  11.) 

Uric  acid  and  its  salts  have,  in  some  degree,  the  power 
of  reducing  copper  compounds  in  an  alkaline  solution  and 
thus  give  with  Fehling's  or  Trommer's  test  results  which 
are  similar  to  those  obtained  with  glucose.  When  the  dry 
substance  is  warmed  with  nitric  acid  it  is  oxidized,  and 


URIC   ACID.  163 

then   gives   with   ammonia   a   reddish-purple   salt,   which 
serves  to  detect  and  identify  the  acid. 

The  urates  as  found  in  the  urine  are  either  in  solu- 
tion or  form  a  sediment.  The  latter  is  generally  amor- 
phous and  is  always  colored  yellow  to  brown.  Acid  sodium 
urate  may  occur  in  spherical  aggregations  of  microscopic 
acicular  crystals.  Ammonium  urate,  formed  when  urine 
becomes  alkaline  by  fermentation,  may  be  found  as 
brownish  spherules  covered  with  irregular  spicules,  the 
so-called  "thorn-apple"  crystals.  (Plate  II,  9  and  11.) 
The  amount  of  uric  acid  in  urine  is  sometimes  found  by 
precipitating  from  a  measured  volume  of  urine  by  hydro- 
chloric acid,  the  albumin  having  first  been  removed  if  it 
is  present.  After  washing  the  crystals  they  are  weighed. 
The  results  thus  obtained  are  too  low,  because  of  the  slight 
solubility  of  the  crystals  in  water.  Volumetric  methods 
may  also  be  employed.  These  are  as  accurate  and  no  more 
difficult. 

351. — Prepare  uric  acid  from  urine  by  quite  strongly 
acidifying  a  beakerful  with  hydrochloric  acid.  In  twenty- 
four  hours  the  uric  acid  will  have  separated.  Examine  the 
crystals  under  the  microscope.  It  can  be  purified  and 
gradually  freed  from  the  color  which  it  derives  from  the 
urine  by  repeatedly  dissolving  in  concentrated  sulphuric 
acid  and  reprecipitating  by  diluting  with  water. 

352. — Precipitate  from  urine  the  uric  acid  with  acids 
of  varying  concentration,  acting  for  different  times.  Sketch 
the  principal  forms  obtained. 

353. — Dissolve  a  few  of  the  crystals  of  the  acid  in 
sodium  hydrate  and  add  a  few  drops  of  Fehling's  solution. 
Boil  and  the  red  cuprous  oxid  will  be  formed,  best  seen 
by  the  use  of  a  dark  background. 


164  THE    URINE. 

354. — To  a  small  quantity  of  uric  acid  in  a  porcelain 
dish  add  a  few  drops  of  dilute  nitric  acid  and  evaporate 
to  dryness,  holding  the  dish  over  a  small  flame  with  the 
hand  in  order  to  avoid  heating  too  highly.  A  reddish-yel- 
low residue  is  left.  Pour  into  the  dish  a  drop  of  ammonia 
without  at  first  letting  it  come  directly  into  contact  with 
the  residue.  In  a  short  time  the  residue  becomes  colored 
reddish  purple.  The  ammonia  may  be  added  directly  to 
the  residue  if  an  excess  is  not  used.  An  excess  destroys 
the  color.  The  addition  of  a  drop  of'  sodium  hydrate 
changes  the  color  to  a  bluish  purple,  which  is  destroyed  on 
warming.  The  test  is  called  the  murexid  test. 

355.  PREPARATION  OF  AMORPHOUS  ACID  URATES. — Dissolve 
uric  acid  in  a  slight  excess  of  sodium  hydrate,  and  then  pass  carbon 
dioxid  into  the  cold  solution  until  it  is  saturated.  Acid  sodium 
urate  separates  in  amorphous  masses. 

356. — Test  the  solubility  of  the  acid  sodium  urate  by 
warming  with  a  small  quantity  of  water.  It  will  dissolve, 
and,  if  not  too  much  water  has  been  used,  will  separate  out 
again  when  it  cools. 

357. — Prepare  crystallized  acid  urates  by  dissolving  a 
little  uric  acid  in  a  warm  solution  of  sodium  phosphate. 
Filter,  if  necessary,  and  allow  the  filtrate  to  stand  and 
evaporate.  The  sodium  urate  will  crystallize  as  masses  of 
acicular  crystals. 

358.  FOMN'S  METHOD  FOR  THE  QUANTITATIVE  DETERMINA- 
TION OF  URIC  Acnx — Prepare  a  solution  in  water,  one  liter  of 
which  shall  contain  500  grammes  of  ammonium  sulphate,  5 
grammes  of  uranium  acetate  and  6  cubic  centimeters  of  glacial 
acetic  acid.  Of  this  add  75  cubic  centimeters  to  300  cubic  centi- 
meters of  urine  in  a  500  cubic  centimeter  flask,  mix,  and  after  five 
minutes  filter  through  a  plaited  filter.  Take  two  portions  of  the 


HIPPURIC   ACID.  165 

filtrate,  125  cubic  centimeters  each,  pour  them  into  beakers,  add 
5  cubic  centimeters  of  concentrated  ammonia  and  let  the  precipi- 
tated urates  stand  until  the  next  day.  Filter  and  wash  the  urates, 
using  a  10-per-cent.  ammonium  sulphate  solution  for  this  as  well 
as  for  transferring  to  the  filter.  Then  spread  out  the  paper  and 
wash  off  the  precipitates  into  beakers,  using  about  100  cubic  centi- 
meters of  water  for  each.  Add  15  cubic  centimeters  of  concen- 
trated sulphuric  acid  and  titrate  immediately  with  one-twentieth 
normal  potassium  permanganate,  containing  1.57  grammes  per 
liter,  stopping  when  the  solution  is  first  pink  throughout.  For 
each  cubic  centimeter  of  permanganate  0.00375  gramme  of  uric 
acid  has  been  oxidized.  Calculate  the  amount  in  the  urine,  adding 
a  correction  of  0.003  gramme  for  every  100  cubic  centimeters  of 
urine  employed  because  of  the  solubility  of  the  urates. 

Heintz's  Method  for  the  quantitative  determination  of  uric 
acid  is  less  accurate.  It  consists  in  adding  to  200  cc.  of  urine 
which  contains  no  albumin  or  sugar  20  cc.  of  concentrated  hydro- 
chloric acid;  the  precipitated  crystals  are  collected  on  a  weighed 
filter,  washed  with  water  until  the  drop  of  the  filtrate  shows  no 
chlorin  reaction  with  a  drop  of  silver  nitrate  and  nitric  acid,  then 
washed  with  alcohol.  After  drying  three  hours  at  110°  the  uric 
acid  is  to  be  rapidly  weighed  on  the  paper.  It  is  customary  to 
add  to  the  weight  0.00038  grm.  for  each  10  cc.  of  filtrate  and  wash 
water  as  a  correction  for  the  acid  which  is  dissolved. 


HIPPURIC  ACID  (C6H5COHNCH2C02H). 

This  occurs  normally  in  the  urine,  but  in  that  of 
human  beings  only  in  very  small  quantities.  It  is  found 
here  in  larger  amounts  after  the  internal  use  of  benzoic 
acid.  It  increases  with  a  vegetable  diet  and  is  abundant 
in  the  urine  of  herbivorous  animals.  It  forms  translucent, 
four-sided  prisms,  somewhat  soluble  in  water.  The  acid 
can  be  made  synthetically  by  heating  benzoic  anhydrid, 
(C6HBCO)20,  with  glycocoll,  NH2CH2C02H:— 

(C6H5CO)20  +  2NH2CH2C02H  =  2C6H5CONHCH2CO2H  +H2O  . 


166  THE   URINE. 

When  hippuric  acid  is  heated  with  mineral  acids  or  alka- 
lies it  decomposes  again  into  glycocoll  and  benzoic  acid. 

359.  PREPARATION  OF  HIPPURIC  ACID. — Take  inter- 
nally 2  grammes  of  pure  sodium  benzoate  and  collect  the 
urine  for  the  next  twenty-four  hours.  Make  it  strongly 
alkaline  with  milk  of  lime.  Warm,  filter,  and  evaporate 
the  filtrate  to  a  syrup  on  the  water-bath.  After  it  has 
cooled  acidify  strongly  with  concentrated  hydrochloric  acid. 
Stir  and  filter,  washing  with  a  little  very  cold  water.  Dis- 
solve the  crystals  in  the  smallest  possible  amount  of  boiling 
water.  To  destroy  the  coloring  matter  pass  chlorin  gas 
into  the  hot  solution  until  it  is  light  yellow.  Then  cool 
it,  filter,  and  wash  the  crystals  with  a  very  little  cold  water. 
If  they  are  still  colored  they  can  be  still  further  purified 
by  dissolving  in  water  and  boiling  with  a  little  animal 
charcoal.  Filter,  and  let  the  acid  crystallize  from  the 
filtrate.  Sketch  the  crystals  and  hand  in  sketches. 

360. — Heat  a  few  of  the  dry  crystals  in  a  glass  tube. 
They  melt  and  turn  red,  then  give,  at  first,  a  hay-like  odor, 
afterward  the  odor  of  bitter  almonds,  from  the  hydrocyanic 
acid  formed.  On  the  cooler  part  of  the  tube  is  a  sublimate 
of  benzoic  acid. 

361. — On  a  few  crystals  in  a  test-tube  pour  about  a 
cubic  centimeter  of  concentrated  nitric  acid,  and  bring  to 
a  boil.  Evaporate  to  dryness  in  a  porcelain  dish  on  a 
water-bath.  The  residue,  when  heated  in  a  dry  glass  tube, 
gives  the  odor  of  bitter  almonds.  This  test  can  be  used  to 
detect  small  quantities  of  hippuric  acid. 

362.  SEPARATION  OF  GLYCOCOLL,  FROM  HIPPURIC  ACID. — 
Boil  in  a  flask  1  part  of  the  acid  for  ten  to  twelve  hours  with  4 
parts  of  dilute  sulphuric  acid.  Use  an  inverted  condenser  to  pre- 
vent evaporation.  Let  the  liquid  cool  and  filter  out  the  benzoic 
acid.  Concentrate  the  nitrate  and  mix  it  in  a  separatory  funnel 


CREATININ.  CHLORIDS.  167 

with  ether,  which  removes  the  last  traces  of  benzoic  acid.  Sepa- 
rate the  aqueous  solution  of  glycocoll  from  the  ether,  add  to  it 
barium  carbonate  until  the  sulphuric  acid  has  been  neutralized, 
filter,  wash  and  evaporate  the  nitrate  until  the  glycocoll  com- 
mences to  crystallize.  It  has  a  sweet  taste,  whence  its  name. 


CREATININ. 

363. — To  separate  the  creatinin  from  urine  add  to  100  cubic 
centimeters  of  the  latter  5  to  6  cubic  centimeters  of  saturated  solu- 
tion of  sodium  acetate,  then  20  to  25  cubic  centimeters  of  a 
saturated  solution  of  mercuric  chlorid.  The  precipitated  sul- 
phates, phosphates  and  urates  are  now  to  be  filtered  out  and  the 
filtrate  allowed  to  stand  for  twenty-four  hours.  There  is  a 
separation  of  the  creatinin  combined  with  mercury  as  transparent 
globules.  Make  sketches  of  its  appearance  under  the  microscope. 


CHLORIDS. 

In  the  urine  the  excreted  chlorin,  of  which  there  is 
normally  in  a  day  6  to  10  grammes,  is  united  principally 
with  sodium.  There  is  a  small  part  with  potassium  as 
potassium  chlorid.  The  excretion  of  chlorids  in  health  is 
increased  with  salt  food  and  with  large  quantities  of  drink. 
Chlorids  are  necessary  in  the  fluids  of  the  body'  for  the 
proper  performance  of  their  functions.  When  more  chlorin 
is  required  by  the  body  the  chlorids  are  held  back  by  the 
kidneys  from  the  urine.  When  there  is  a  less  demand  in 
the  body  the  kidneys  excrete  the  chlorids.  Thus,  in  pneu- 
monia and  other  diseases,  where  there  are  serous  exuda- 
tions, the  chlorids  are  withdrawn  from  the  circulation  to 
form  the  constituents  of  these  fluids,  as  is  shown  by  their 
decrease  in  the  urine.  When  the  pathological  exudations 
are  absorbed  the  amount  of  urinary  chlorids  increases.  In 
fevers  there  is  a  decrease  in  the  chlorids  of  the  urine  until 


168  THE   URINE. 

the  crisis,  then  an  increase.  In  chronic  diseases  the  amount 
of  chlorin  gives  some  indication  of  the  digestive  power, 
6  to  10  grammes  per  day  being  normal,  and  less  than  5 
grammes  daily  showing  weakness  of  digestion,  providing 
that  an  excessive  amount  has  not  been  removed  by  other 
means,  like  serous  exudations  or  diarrhoeic  discharges.  An 
excessive  excretion  of  chlorin  (15  to  20  grammes  daily) 
is  found  in  diabetes  insipidus.  In  dropsical  conditions  it 
is  a  favorable  sign,  showing  the  absorption  of  the  fluid. 
The  quantity  of  chlorin  can  be  determined  by  ascer- 
taining how  much  silver  nitrate  is  required  to  precipitate 
it. 

NaCl  +  AgN03  =  AgCl  +  NaN03. 

58.4  parts  170  parts 

To  ascertain  when  the  chlorin  has  all  united  with  the 
silver  a  little  yellow  potassium  chromate  is  added.  The 
silver  forms  first  a  white  silver  chlorid,  and  when  the  chlo- 
rin has  been  precipitated  it  forms  the  red  silver  chromate. 

364. — Acidify  a  portion  of  urine  in  a  test-tube  with 
nitric  acid  and  add  a  little  silver  nitrate.  A  white  precipi- 
tate which  turns  dark  in  the  sunlight  indicates  the  pres- 
ence of  chlorids. 

365.  DETERMINATION  OF  QUANTITY  or  CHLORIN  IN 
URINE. — For  clinical  purposes  the  following  method  is  suf- 
ficiently accurate :  Measure  with  a  pipette  10  cubic  centi- 
meters of  urine  and  dilute  with  about  100  cubic  centi- 
meters of  water.  Add  a  few  drops  of  yellow  potassium 
chromate  solution ;  then  allow  to  flow  into  it  from  a  burette 
a  solution  which  contains  17.000  grammes  of  fused  silver 
nitrate  in  a  liter.  As  soon  as  the  color  of  the  precipitate 
changes  from  white  to  reddish,  read  off  the  volume  of  silver 
solution  which  has  been  used.  Each  cubic  centimeter  of 


PHOSPHATES.  169 

this  will  precipitate  0.00354  gramme  of  chlorin,  equal  to 
0.00584  gramme  of  sodium  chlorid.  Calculate  the  per- 
centage of  chlorin  by  weight  in  the  urine.  The  change  of 
color  from  white  to  red  can  be  more  plainly  seen  by  yellow 
light  (gaslight)  than  by  daylight.  There  are  present  in 
the  urine  some  other  substances  which  are  precipitated  by 
silver  nitrate  like  the  chlorin.  To  make  approximate  cor- 
rection for  these,  1  cubic  centimeter  may  be  subtracted 
from  the  number  used. 

366. — A  more  accurate  result  can  be  obtained  if  the  organic 
matter  is  first  destroyed.  To  10  cubic  centimeters  of  urine  in  a 
thin  porcelain  or  platinum  dish  add  about  1  gramme  of  sodium 
nitrate  and  2  grammes  of  potassium  nitrate,  both  free  from 
chlorids.  Evaporate  to  dryness  and  carefully  heat  to  fusion. 
Cool,  dissolve  in  water,  slightly  acidify  with  nitric  acid,  then  make 
exactly  neutral  with  sodium  carbonate  and  titrate  with  silver 
nitrate,  calculating  the  amount  of  chlorin  as  in  the  preceding 
experiment. 

PHOSPHATES. 

The  phosphoric  acid  of  the  urine  is  united  with  two 
classes  of  bases:  the  alkalies, — sodium  and  potassium, — 
and  the  alkaline  earths, — calcium  and  magnesium.  The 
compounds  are  called,  respectively,  "alkaline"  and  "earthy" 
phosphates.  The  alkaline  phosphates  are  soluble  in  water. 
The  earthy  phosphates  are  insoluble  in  water  or  alkalies, 
but  are  dissolved  by  acids.  They  consequently  appear  in 
the  urine  in  the  insoluble  form  whenever  it  becomes  alka- 
line, either  by  fermentation  or  by  the  addition  of  reagents. 
They  may  also  be  precipitated  by  boiling.  The  amorphous 
white  precipitate  thus  obtained  is  often  mistaken  for  albu- 
min. It  can  be  distinguished  by  being  easily  soluble  in 
acids,  which  is  not  the  cr.se  with  albumin.  "When  ammonia 
is  present,  as  in  fermentation,  the  magnesium  forms  an 


170  THE   URINE. 

insoluble  salt  with  two  bases,  NH4MgP04.  In  urinary 
analysis  it  is  referred  to  as  triple  phosphate.  It  is  crystal- 
line, sometimes  in  the  form  of  snow-flakes,  but  more  com- 
monly in  prismatic  crystals  often  spoken  of  as  "coffin-lid 
crystals,"  from  their  supposed  resemblance  to  the  lid  of  a 
coffin.  (Plate  II,  8.) 

The  phosphoric  acid  of  the  urine  is  mainly  that  taken 
in  the  food,  but  a  part  conies  from  the  oxidized  phos- 
phorus compounds  of  the  tissues,  such  as  lecithin  and  the 
nuclein  compounds.  The  presence  of  a  sediment  of  the 
earthy  phosphates  shows  simply  that  the  urine  is  alkaline, 
and  is  no  indication  that  an  excessive  amount  is  being  ex- 
creted. Animal  foods  are  richer  in  phosphoric  acid  com- 
pounds than  vegetable;  hence  with  these  we  find  more  in 
the  urine. 

Experience  has  shown  that  there  is  a  diminution  of 
the  excreted  phosphoric  acid  in  many  pathological  condi- 
tions. This  is  true  in  most  acute  infectious  diseases,  in 
nephritis,  gout,  and  rheumatism.  In  diabetes  mellitus 
there  is  an  increase.  Still,  with  the  exception  of  the  bones, 
the  tissues  of  the  body  contain  but  comparatively  small 
amounts  of  phosphorus  compounds,  and  with  our  present 
knowledge  it  is  difficult  to  draw  definite  conclusions  re- 
garding the  decomposition  of  such  tissues  from  the  varia- 
tions in  the  eliminated  phosphoric  acid. 

367. — Make  a  specimen  of  urine  alkaline  with  sodium 
hydrate.  The  earthy  phosphates  are  precipitated  in  an 
amorphous  form.  Examine  under  the  microscope.  See 
that  they  are  dissolved  again  by  acidifying  with  even  a 
weak  acid,  like  acetic. 

3G8. — Filter  out  the  earthy  phosphates  and  test  the 
filtrate  for  the  phosphoric  acid  of  the  alkaline  phosphates 


PHOSPHATES.  171 

by  adding  magnesia  mixture.  (This  is  magnesium  sul- 
phate made  alkaline  with  ammonia  and  enough  ammonium 
chlorid  to  dissolve  the  precipitate  first  formed.)  With 
phosphoric  acid  or  its  salts  it  gives  a  white  crystalline 
precipitate. 

369. — Form  triple  phosphate  by  making  urine  faintly 
alkaline  with  ammonia  and  allowing  it  to  stand  until  the 
precipitate  settles.  Examine  under  the  microscope  for  the 
"coffin-lid"  crystals.  They  can  be  more  abundantly  formed 
for  microscopic  examination  by  adding  to  the  urine  a  little 
of  a  solution  of  magnesium  sulphate  before  making  it  alka- 
line with  ammonia. 

370.  DETERMINATION  OF  AMOUNT  OF  PHOSPHORIC  ACID. — 
Prepare  the  following  solutions:  — 

1.  Uranium  acetate:    Dissolve  about  34  grammes  of  crystal- 
lized uranium  acetate  in  water  and  dilute  to  one  liter.     This  solu- 
tion will  be  a  little  too  strong.     Its  exact  strength  must  be  found 
by  the  method  to  be  described  later. 

2.  A  solution  of  Na2HP04,  12H20  (crystallized  disodium  phos- 
phate), one  liter  of  which  shall  contain  10.085  grammes  of  the  pure 
crystallized  salt.     This  salt  gives  up  its  water  of  crystallization 
when  exposed  to  the  air,  and  cannot  then  be  used.     The  crystals 
must  be  perfectly  bright.     Fifty  cubic  centimeters  of  the  solution 
contain  0.1  gramme  of  P2OB  (phosphoric  anhydrid). 

3.  Solution  of  sodium  acetate  of  which  one  liter  contains  100 
cubic  centimeters  of  30-per-cent.  acetic  acid  and   100  grammes  of 
sodium  acetate. 

4.  Solution  of  cochineal  made  by  digesting  for  some  time  1 
gramme   of  powdered   cochineal   in   a   mixture   of  20   volumes   of 
alcohol  \vith  60  volumes  of  water.    Filter  or  decant  the  liquid. 

Operation. — First  ascertain  the  strength  of  the  uranium  solu- 
tion. To  accomplish  this,  measure  with  a  pipette  50  cubic  centi- 
meters of  the  sodium  phosphate  solution  into  a  beaker;  add  5 
cubic  centimeters  of  the  sodium  acetate  solution  and  a  few  drops 
of  cochineal.  Heat  to  boiling,  and  then  from  a  burette  run  in  the 


172  THE   UEINE. 

uranium  solution,  drop  by  drop,  until  a  greenish  color  is  produced. 
The  phosphoric  acid  has  then  been  precipitated.  Since  1  cubic 
centimeter  of  the  uranium  solution  ought  to  precipitate  0.005 
gramme  of  P206,  exactly  20  cubic  centimeters  should  have  been 
used  for  the  50  cubic  centimeters  of  sodium  phosphate.  If  this  is 
not  the  quantity  which  has  been  used,  first  ascertain  accu- 
rately how  much  is  needed  and  then  dilute  the  uranium  solution 
so  that  1  cubic  centimeter  precipitates  0.005  gramme  of  P2O6.  If, 
for  instance,  17.5  cubic  centimeters  have  been  used  instead  of  20 
cubic  centimeters  there  must  be  added  2.5  cubic  centimeters  of 
water  for  every  17.5  cubic  centimeters  of  the  uranium  solution. 

The  amount  of  P2O6  in  urine  can  now  be  determined  in  the 
same  manner,  using  urine  instead  of  the  sodium  phosphate  solu- 
tion. Calculate  the  percentage  of  P2O6  present,  knowing  that  there 
is  0.005  gramme  for  each  cubic  centimeter  of  uranium  solution 
which  has  been  used. 

If  it  desired  to  determine  the  amount  of  phosphoric  acid 
combined  with  the  alkaline  earths  (calcium  and  magnesium),  as 
distinguished  from  the  phosphates  of  sodium  and  potassium,  the 
former  class  can  be  precipitated  from  200  cubic  centimeters  of 
urine  by  ammonium  hydrate  and,  after  settling,  can  be  removed 
by  nitration.  Wash  the  precipitate  well  with  ammonium  hydrate, 
then  pierce  the  filter  and  rinse  the  precipitate  by  a  jet  of  water 
into  a  beaker.  Dissolve  it  in  as  small  a  quantity  of  acetic  acid  as 
possible,  add  5  cubic  centimeters  of  the  sodium  acetate  solution, 
dilute  to  50  cubic  centimeters  and  titrate  as  in  the  preceding 
operation.  This  gives  the  P2O6  which  was  united  with  the  calcium 
and  magnesium.  If  the  total  has  been  determined,  the  difference 
represents  the  phosphoric  acid  originally  combined  with  sodium 
and  potassium. 

SULPHATES. 

The  sulphates  of  the  urine  are  of  two  classes:  (1) 
those  of  which  the  base  is  a  metal,  like  K2S04  and  Na^SO^ 
and  (2)  those  in  which  a  part  or  the  whole  of  the  base  has 
been  replaced  by  an  organic  radical,  like  KC6H5S04. 


SULPHATES.  173 

Those  of  the  first  class  are  called  the  inorganic,  and  the 
second  the  organic,  or  ethereal,  sulphates.  The  latter  dif- 
fer from  the  inorganic  in  not  forming  an  insoluble  precipi- 
tate upon  the  addition  of  a  barium  salt  as  the  inorganic 
do.  The  two  classes  can  be  separated  by  this  means.  After 
the  removal  of  the  inorganic  sulphuric  acid  by  barium 
chlorid  the  organic  sulphates  can  be  decomposed  by  means 
of  boiling  hydrochloric  acid: — 

KC6H5S04  +.H20  ==  C6HBOH  +  KHS04. 

The  acid  will  then  give  the  white  precipitate  of  barium 
sulphate  if  barium  chlorid  be  added. 

The  total  amount  of  combined  sulphuric  acid  excreted 
by  an  adult  in  twenty-four  hours  is  2  to  3  grammes.  It 
is  derived  partly  from  that  already  formed  in  the  food, 
which  passes  without  change  into  the  urine,  but,  for  the 
most  part,  from  the  oxidation  of  sulphur  compounds,  like 
albumin,  in  the  body.  Variations  in  the  total  sulphuric 
acid  in  general  indicate  the  rate  of  oxidation  of  sulphur 
compounds.  It  is  increased  by  taking*  such  compounds, 
e.g.,  by  a  meat  diet.  It  is  decreased  by  a  vegetable  diet. 

The  organic  sulphates  normally  make  up  about 
one-tenth  of  the  total  sulphates.  The  organic  bases  of 
these  are  such  compounds  as  phenol  (C6H5OH),  cresol 
(C6H4CH3OH),  indoxyl  (C8H6NOH),  etc.  These  bases  are 
formed  by  the  putrefaction  of  albuminous  substances;  con- 
sequently, when  such  putrefaction  is  in  progress  in  the 
body  the  organic  sulphates  increase  in  the  urine.  They 
may  be  formed  in  the  intestine  or  absorbed  from  some 
other  source. '  In  the  former  case  they  are  increased  when- 
ever there  is  a  serious  stoppage  of  the  food,  as  in  ileus  or 
in  peritonitis  with  atony  of  the  intestine.  In  ordinary  con- 
stipation there  is  no  marked  increase.  In  diseases  which 


174  THE   URINE. 

are  accompanied  by  an  internal  formation  of  pus  there  is  an 
increased  amount  of  organic  sulphates  in  the  urine,  and  this 
fact  may  be  used  to  judge  whether  the  pus-forming  stage 
has  been  reached.  This  is  the  case  in  foetid  bronchitis, 
carcinoma  of  the  stomach  or  intestine,  diphtheria,  pyaemia, 
etc.  If  the  formation  is  from  putrefaction  in  the  intestine 
it  will  be  diminished  by  taking  antiseptic  remedies,  like 
calomel,  or  those  which,  by  their  purgative  action,  remove 
the  contents  of  the  intestine  before,  this  putrefaction  has 
occurred. 

The  compound  of  indol  which  is  found  in  the  urine 
goes  by  the  name  of  indican.    The  indol, 

C6H4  —  CH 


—  CH 

formed  by  the  putrefaction  of  albuminous  substances,  is 
oxidized  after  it  has  been  absorbed  from  the  intestine  or 
elsewhere  in  the  body  and  becomes  indoxyl:  — 

C6H4  —  COH 

I          II 
HN  —  CH 

This  unites  with  potassium  and  sulphuric  acid  to  form 
indican:  — 

C6H4  —  C  —  S04K 


—  CH 

Indican  may  be  easily  oxidized  by  chlorin  or  other  oxidiz- 
ing agents,  and  then  forms  indigo  blue:  — 

C6H4  —  CO    CO  —  C6H4 

I          I         I  I 

—  C  =  C    —  NH 


ORGANIC   SULPHATES.  175 

Putrefaction  of  nitrogenous  compounds  in  the  small  in- 
testine seems  to  be  more  productive  of  indican  than  when 
it  goes  on  in  the  large  intestine.  Sometimes  the  indican  is 
decomposed  in  the  urine,  the  indigo  being  set  free  in  the 
form  of  blue  or  red  microscopic  crystals.  It  is  usually  dis- 
solved as  a  sulphate,  however,  until  the  indigo  is  formed 
by  an  oxidizing  agent.  It  is  normally  present  in  large 
quantities  in  the  urine  of  the  horse,  where,  because  of  the 
long  intestine,  the  residue  from  the  food  requires  a  con- 
siderable time  to  pass  from  the  body. 

371.  PREPARATION  OF  POTASSIUM  PHENOL  SULPHATE 
(KC6H5SO4). — First  prepare,  if  it  is  not  at  hand,  potassium  pyro- 
sulphate  by  mixing  25  grammes  of  finely-powdered  potassium 
sulphate  with  15  grammes  of  concentrated  sulphuric  acid,  then 
heating  (best  in  platinum  dish).  The  heating  should  be  done 
under  a  hood,  to  avoid  the  acid  fumes.  The  heat  should  be 
gently  applied  at  first,  stirring  until  all  the  crystals  have  dissolved. 
When  it  ceases  to  bubble  increase  the  heat  to  low  redness.  Allow 
it  to  cool,  but  before  it  solidifies  it  is  best  to  carefully  pour  it  upon 
a  piece  of  clean  sheet  iron.  Powder  finely  the  potassium  pyro- 
sulphate  (K2S2O7)  thus  obtained. 

In  a,  thin  glass  flask  holding  about  a  liter  dissolve  15  grammes 
of  potassium  hydrate  in  20  or  25  cubic  centimeters  of  water,  then 
add  25  grammes  of  crystallized  phenol  (carbolic  acid,  CfiH5OH). 
When  it  has  dissolved  let  it  cool  to  60°  or  70°  C.,  and,  while  stir- 
ring well,  add  gradually  in  small  quantities  30  grammes  of  potas- 
sium pyrosulphate  powdered  as  finely  as  possible.  Keep  it  at  a 
temperature  of  "from  60°  to  70°  for  from  eight  to  ten  hours,  shak- 
ing often.  Then  add  about  125  cubic  centimeters  of  boiling  95- 
per-cent.  alcohol,  and  filter  while  it  is  hot.  This  filtration  is  best 
performed  in  a  hot-water  funnel — that  is,  one  which  is  surrounded 
with  a  hot-water  jacket.  Otherwise  the  salt  will  crystallize  out 
before  the  liquid  has  passed  through  the  filter.  As  soon  as  the 
filtrate  cools,  the  potassium  phenyl  sulphate  crystallizes  in  pearly 
plates.  It  should  be  filtered  out  and  recrystallized  from  a  small 
quantity  of  boiling  alcohol. 


176  THE   URINE. 

372. — Test  a  solution  of  this  organic  sulphate  with 
barium  chlorid.  There  is  no  precipitate.  Compare  the 
result  with  that  obtained  from  an  inorganic  sulphate,  like 
magnesium  sulphate,  with  barium  chlorid. 

373. — Acidify  a  solution  of  an  organic  sulphate  with 
hydrochloric  acid,  boil,  and  add  barium  chlorid.  The  acid 
has  decomposed  the  sulphate;  so  that  a  precipitate  of  ba- 
rium sulphate  is  now  obtained. 

374. — Show  that  a  mixture  of  the  two  classes  of  sul- 
phates, as  in  urine,  can  be  detected  in  this  way.  First 
acidify  by  acetic  acid,  then,  after  adding  barium  chlorid, 
let  the  test-tube  stand  at  least  half  an  hour  in  a  beaker  of 
boiling  water.  The  inorganic  sulphates  are  thus  precipi- 
tated as  barium  sulphate,  but  not  the  organic.  Filter,  and 
test  the  nitrate  with  a  drop  of  barium  chlorid.  If  enough 
was  added  at  first  there  will  be  no  precipitate.  If  there  is, 
more  barium  chlorid  must  be  used,  and  the  heating  re- 
peated. When  the  filtrate  remains  clear,  acidify  with  hy- 
drochloric acid  and  boil.  The  precipitate  is  from  the  de- 
composed organic  sulphates  united  with  the  barium  chlorid 
previously  added. 

375. — Insert  into  a  rabbit's  stomach  a  wide,  flexible 
catheter  or  rubber  tubing,  passing  it  through  a  short  piece 
of  glass  tubing  held  between  the  animal's  teeth.  Intro- 
duce by  this  tube  a  gramme  of  ortho-nitro-phenyl-propiolic 
acid  and  collect  the  urine  for  twenty-four  hours.  It  will 
contain  a  large  quantity  of  indican.  Eead  the  literature 
on  the  relationship  of  the  above  acid  to  indigo,  and  explain 
the  formation  of  indican. 

376. — Test  urine  for  indican  by  adding  to  half  a  test- 
tubeful  an  equal  volume  of  concentrated  HC1.  Then  add 
a  minute  fragment  of  calcium  hypochlorite  ("chlorinated 
lime")  and  a  few  drops  of  chloroform  and  shake  gently. 


INDICAN.  177 

Let  the  chloroform  settle  to  the  bottom.  If  indican  is  pres- 
ent in  the  urine  it  will  be  thus  oxidized  to  indigo  blue,  and 
this  colors  the  chloroform.  A  second  piece  of  the  hypo- 
chlorite  may  be  added  and  the  shaking  repeated.  An  ex- 
cess will  destroy  the  blue  color.  Instead  of  calcium  hypo- 
chlorite,  a  few  drops  of  chlorin  water,  bromin  water,  or 
hydrogen  dioxid  can  be  used.  This  is  Jaffe's  test.  . 

377. — Obermayer's  reagent  for  indican  contains  2  to 
4  grammes  of  ferric  chlorid  in  a  liter  of  concentrated  hy- 
drochloric acid.  An  excess  does  not  destroy  the  indigo. 
Mix  equal  volumes  of  the  reagent  and  urine  and  shake. 
Indican  is  oxidized  to  indigo-blue.  This  can  be  taken  up 
by  a  drop  of  chloroform,  as  in  the  preceding  test. 

After  the  internal  use  of  iodin  compounds  the  iodin  is  ex- 
creted largely  through  the  urine.  The  reagents  of  either  Jaffe's 
or  Obermayer's  tests  will  set  it  free  and  it  will  dissolve  in  the 
chloroform  with  a  color  which  simulates  or  conceals  the  indican 
reaction.  A  few  drops  of  sodium  thio-sulphate  solution  will,  how- 
ever, destroy  the  iodin  color,  but  not  that  of  indican. 

378. — Add  to  10  cubic  centimeters  of  urine  a  few  drops  of  a 
solution  of  potassium  iodid  and  make  Jaffa's  or  Obermayer's  test, 
following  this  with  sodium  thio-sulphate. 

379.  THE  QUANTITATIVE  DETERMINATION  OP  URINARY 
INDICAN:  WANG'S  METHOD. — This  depends  upon  the  conversion 
of  indican  to  indigo  by  Obermayer's  reagent  and,  after  extracting 
it  with  chloroform,  determining  its  amount  by  titration  with 
standard  potassium  permanganate.  The  quantity  of  urine  em- 
ployed for  each  test  should  be  modified  according  to  the  amount 
of  indican  as  revealed  by  a  preliminary  test,  50  cubic  centimeters 
being  enough  when  much  is  present  and  250  or  more  when  very 
little  is  found. 

If  the  urine  is  alkaline  acidify  with  acetic  acid  and  precipi- 
tate with  a  20-per-cent.  solution  of  basic  lead  acetate,  avoiding  an 
excess.  Find  the  volume  of  this  mixture,  filter,  measure  off  most 
of  the  filtrate  and  to  it  add  as  much  of  Obermayer's  reagent. 
Shake  this  with  renewed  portions  of  chloroform  until  the  latter  is 

12 


178  THE    URINE. 

not  colored,  separating  the  two  liquids  by  means  of  a  glass-stop- 
pered funnel.  Distill  the  chloroform  from  its  solution,  dry  the 
residue  on  a  water-bath  and  wash  it  with  hot  water,  passing  this 
through  a  small  filter  to  avoid  the  loss  of  indigo.  Dry  the  paper, 
extract  it  with  warm  chloroform,  add  this  to  the  washed  residue 
and  distill  off  the  chloroform.  Warm  the  indigo  on  a  water-bath 
for  five  to  ten  minutes  with  concentrated  sulphuric  acid  and  pour 
it  into  .about  100  cubic  centimeters  of  water,  rinsing  the  vessel 
with  a  little  more  water.  Filter  and  titrate  with  a  standard 
solution  of  potassium  permanganate  until  the  color  is  colorless  or 
yellowish. 

The  permanganate  contains  about  0.1  gram  to  the  liter. 
It  must  be  frequently  standardized  against  a  solution  of  oxalic  of 
known  strength.  The  indigo  blue  corresponding  to  each  cubic 
centimeter  is  1.04  times  the  weight  of  the  oxalic  acid  used.  From 
this  the  weight  of  indican  can  be  calculated. 


ALBUMINOUS  COMPOUNDS  OF  THE  URINE. 

The  principal  albuminous  substance  occurring  in  the 
urine  is  serum-albumin.  Besides  this  there  may  be  found 
there  serum-globulin,  albumose,  fibrin,  and  possibly  pep- 
tones. The  nucleoalbumins  also  are  not  uncommon,  being 
often  mistaken  for  mucin. 

ALBUMINURIA. 

Serum-albumin  may  find  its  way  into  the  urine  either 
from  the  kidneys  (renal  albuminuria)  or  from  serous 
liquids,- — like  blood,  pus,  or  lymph, — mixing  with  it  at 
some  point  in  the  urinary  tract  below  the  kidneys.  When 
it  is  due  to  degenerative  changes  in  the  kidney  it  is  usually 
accompanied  by  epithelium  from  the  tubules,  often  in  the 
form  of  cylinders  or  casts.  Changes  in  the  composition 
of  the  blood  or  in  the  blood-pressure  may  allow  albumin 
to  pass  through  the  kidney.  This  is  seen  in  anaemic  con- 


ALBUMINURIA.  179 

ditions,  after  some  poisons,  and  in  some  infectious  dis- 
eases, the  kidneys  in  any  of  these  cases  not  being  neces- 
sarily in  a  pathological  state.  Severe  muscular  labor  may 
cause  the  temporary  appearance  of  albumin.  The  quantity 
present  varies  greatly  under  different  conditions,  and  is 
not  necessarily  a  measure  of  the  severity  of  the  disease. 
Still  comparative  tests  in  the  same  case  will  indicate  some- 
thing of  its  progress. 

The  amount  of  albumin  in  the  urine  can  be  deter- 
mined accurately  by  precipitating,  drying,  and  weighing, 
but  the  process  is  a  long  one  for  clinical  purposes.  For  a 
practical  test,  sufficient  to  show  the  variation  in  amount, 
Esbach's  method  can  be  used.  This  depends  upon  precipi- 
tating the  albumin  with  a  solution  containing  1-per-cent. 
picric  acid  and  2-per-cent.  citric  acid.  The  operation  is 
performed  in  a  graduated  test-tube,  called  an  albuminom- 
eter,  the  height  of  the  precipitate  indicating  its  amount. 
Variations  in  temperature  greatly  affect  the  height  of  the 
precipitate;  consequently  in  comparative  determinations 
the  conditions  of  temperature  must  be  always  the  same. 
The  results  are  most  accurate  when  not  more  than  4 
grammes  of  albumin  are  contained  in  a  liter. 

A  more  accurate  method  for  the  determination  of  the 
amount  of  albumin  in  urine  is  to  weigh  it  after  coagula- 
tion. Filter  it  if  it  is  not  clear,  then  drop  in  two  or  three 
drops  of  very  dilute  acetic  acid  and  heat  to  boiling.  Filter 
on  a  filter  which  has  been  weighed  after  drying  at  100°, 
and  wash  with  warm  water  first,  then  with  alcohol.  Dry 
at  120°  until  its  weight  is  constant.  For  exact  results  the 
precipitate  and  filter  must  be  burned,  and  the  weight  of 
the  ash  subtracted  to  get  the  true  weight  of  the  coagulated 
protein.  If  globulin  is  present  it  will  also  be  found  in  the 
precipitate  with  the  albumin. 


180  THE   URINE. 

TESTS   FOR   ALBUMIN   IN   THE   URINE. 

If  the  urine  is  not  clear  it  must  be  filtered  before 
testing. 

380.  THE  HEAT,  OR  BOILING,  TEST. — Heat  the  urine 
to  boiling  in  a  test-tube,  then  acidify  with  a  few  drops  of 
concentrated  nitric  acid.     If  albumin  is  present,  a  white 
precipitate  remains.    The  earthy  phosphates  precipitate  on 
boiling,  but  are  soluble  in  acids. 

381.  HELLER'S  TEST. — Pour  half  an  inch  of  concen- 
trated nitric  acid  into  a  small  test-tube.    From  a  pipette, 
the  end  of  which  is  held  just  above  the  surface  of  the  acid, 
drop  the  urine  slowly  or  hold  the  tube  in  a  slanting  posi- 
tion and  slowly  pour  upon  the  acid  an  equal  volume  of 
urine.     If  albumin  is  present  a  white  cloud  forms  at  the 
point  of  contact  of  the  two  liquids.    If  the  amount  is  ex- 
ceedingly small,  it  may  not  appear  for  half  an  hour.     (If 
biliary  pigments  are  present  the  ring  may  be  colored.    See 
test  for  these,  Experiment  285.) 

382. — Acidify  2  or  3  cubic  centimeters  of  potassium 
ferrocyanid  solution  with  about  1  cubic  centimeter  of  acetic 
acid,  and  fill  the  test-tube  half  full  of  urine.  Albumin 
gives  a  white,  cloudy  precipitate.  An  excess  of  ferrocyanid 
interferes  with  the  accuracy  of  the  test. 

383. — Add  to  the  urine  in  a  test-tube  about  one-sixth 
of  its  volume  of  a  saturated  solution  of  sodium  chlorid, 
acidify  with  acetic  acid,  and  boil  the  upper  part  of  the 
liquid,  holding  the  tube  by  the  bottom.  Albumin  gives  a 
white  precipitate,  which  shows  plainly  above  the  clear 
liquid  in  the  lower  part  of  the  tube. 

Each  of  these  tests  has  some  objections  to  it  which  must  be 
recognized  in  interpreting  the  results.  By  the  action  of  heat  and 
nitric  acid  some  of  the  albumin  is  decomposed;  hence  the  first  test 


ALBUMIN   TESTS.  181 

is  not  as  sensitive  as  some  others.  This  decomposition  is  greatly 
increased  if  the  urine  is  boiled  after  adding  the  acid.  Besides 
albumin,  there  may  be  precipitated  by  this  test  uric  acid  from  the 
urates  in  very  concentrated  normal  urine,  and  also  resinous  matters 
after  the  administration  of  turpentine  or  the  balsams.  The  resin- 
ous compounds  are  soluble  in  alcohol,  which  does  not  dissolve  albu- 
min. The  uric-acid  compounds  are  colored  instead  of  being  white, 
like  albumin,  and  can  be  filtered  out  and  tested. 

The  ring-test  with  nitric  acid  is  very  sensitive.  It  precipitates 
other  substances  than  albumin, — such  as  the  urates,  mucin,  and 
resinous  substances.  The  urates  do  not  form  a  ring  at  the  plane 
of  contact  of  the  two  liquids,  but  above  it;  and  if  the  urine  is 
previously  diluted  with  two  or  three  times  its  volume  of  water 
they  do  not  appear.  The  resinous  matters  dissolve  in  alcohol.  The 
mucin  precipitate  forms  a  cloud  in  the  upper  part  of  the  liquid 
where  the  acid  is  dilute.  It  dissolves  in  strong  nitric  acid. 

Potassium  ferrocyanid  and  acetic  acid  will  detect  very  small 
quantities  of  albumin.  Albumose  is  also  precipitated  if  present. 
If  the  acid  alone  produces  a  cloudiness  it  is  mucin  or  resinous  com- 
pounds. These  must  be  removed  by  filtration  before  adding  the 
ferrocyanid. 

In  the  sodium  chlorid  and  acetic  acid  test  the  precipitate 
formed  on  boiling  is  acid  albumin,  which  is  insoluble  in  the  salt 
solution.  Resinous  matters  may  be  precipitated,  but  not  mucin. 

384.  DETERMINATION  OF  AMOUNT  OF  ALBUMIN  IN 
URINE  BY  ESBACH'S  METHOD. — The  urine  must  not  have 
a  specific  gravity  above  1.008,  otherwise  it  must  be  diluted. 
If  it  is  not  distinctly  acid  in  reaction  it  must  be  made  so 
by  acetic  acid.  Fill  the  albuminometer  with  urine  to  the 
mark  U.  Add  the  reagent  to  the  mark  R.  Close  with  a 
cork  and  mix  gently,  avoiding  hard  shaking,  which  intro- 
duces air  bubbles  into  the  precipitate  and  thereby  prevents 
its  settling.  Let  it  stand  at  the  temperature  of  the  room 
(60°  to  70°  F.)  for  twenty-four  hours.  The  height  of 
the  precipitate  indicates  the  number  of  grammes  of  albu- 
min per  liter,  or  parts  in  a  thousand. 


182  THE   URINE. 

GLOBULIN,  ALBUMOSE,  AND  PEPTONES. 

Globulin  is  found  in  the  urine  only  with  albumin. 
It  passes  into  the  urine  in  much  the  same  manner,  and 
has  no  especial  diagnostic  value. 

385. — To  10  cubic  centimeters  of  the  clear  urine  (fil- 
tered if  necessary)  add  an  equal  volume  of  a  saturated 
solution  of  ammonium  sulphate.  The  serum  globulin  will 
be  precipitated,  but  not  the  albumin. 

386. — Saturate  the  clear  urine  with  finely  powdered 
magnesium  sulphate  without  warming.  Serum  globulin 
is  precipitated.  This  can  be  removed  by  filtration  and 
confirmed  by  the  usual  tests  for  globulins. 

Albumose  may  be  formed  in  urine  by  bacterial  action 
from  albumin.  It  may  easily  escape  discovery,  since  it  is 
not  coagulated  by  heat.  It  is  often  the  precursor  of  albu- 
min, and,  as  such,  a  knowledge  of  its  presence  is  impor- 
tant. Before  testing  for  its  presence  the  other  albuminous 
substances  must  be  removed.  After  removing  albumin  by 
boiling  the  liquid  slightly  acidified  by  acetic  acid,  albumose 
can  be  detected  by  its  giving  a  precipitate  upon  saturation 
with  sodium  chlorid,  which  dissolves  on  heating  and  re- 
appears on  cooling. 

The  results  of  the  latest  research  have  shown  that 
much  of  what  has  been  regarded  as  peptone  in  urine  is  one 
of  the  albumoses  which  closely  resembles  it,  and  it  is  an 
open  question  whether  peptones  are  ever  found  in  this  ex- 
cretion. Nevertheless  we  may  temporarily  retain  the  name 
peptonuria  for  the  condition,  with  the  understanding  that, 
as  our  knowledge  becomes  greater,  it  may  have  to  be  aban- 
doned. The  peptones  or  albumoses  are  not  normally  found 
in  the  blood,  being  converted  in  the  intestinal  mucous 


PEPTONES.  183 

membrane  into  another  form,  probably  into  an  -albumin. 
When  anything  interferes  with  this  conversion,  or  when 
they  are  otherwise  introduced  into  the  blood,  they  pass  into 
the  urine.  Diseases  of  the  intestine,  like  carcinoma  or 
ulceration,  may  prevent  conversion  to  albumin,  giving  rise 
to  enterogenic  peptonuria.  Peptone  and  albumose  are 
formed  by  the  decomposition  of  albuminous  substances  by 
other  means  than  by  digestion;  as,  for  example,  by  putre- 
faction. Diseases  which  are  characterized  by  a  formation 
of  peptones  are  often  accompanied  by  peptonuria.  This  is 
the  so-called  "pyogenic  peptonuria/'  It  is  found  when 
there  is  much  formation  of  pus  in  a  body-cavity,  as  in 
croupous  pneumonia  and  with  deep-seated  abscesses. 

The  following  tests  can  be  used  with  urine  containing 
a  considerable  peptone : — 

387. — Heat  50  cubic  centimeters  of  urine  to  boiling; 
acidify  if  necessary  with  a  few  drops  of  acetic  acid,  filter- 
ing if  it  precipitates,  and,  while  hot,  add  powdered  am- 
monium sulphate  to  the  filtrate  as  long  as  it  dissolves  and 
until  there  are  some  crystals  in  the  bottom.  Filter  after 
cooling.  This  leaves  the  peptone  in  solution.  To  insure 
complete  precipitation  of  the  other  proteins  the  saturation 
with  ammonium  sulphate  may  have  to  be  repeated.  When 
this  has  been  done  and  no  further  precipitate  results,  test 
portions  of  the  filtrate  with  (1)  tannic  acid  with  twice 
its  volume  of  water;  (2)  potassio-mercuric  iodid.  Each 
should  give  a  yellowish-white  precipitate.  The  biuret 
test  can  be  tried,  but  is  not  as  sensitive  as  the  others.  With 
peptones,  if  no  excess  of  copper  sulphate  is  used,  it  should 
give  a  pink  with  no  shade  of  blue.  A  large  amount  of 
sodium  hydrate  must  be  present. 


184  '  THE    URINE. 


FIBRINURIA. 

Through  haemorrhage  or  exudation  of  serous  fluids 
into  the  urinary  passages  the  urine  sometimes  becomes 
mixed  with  fibrinogen,  and  this  may  form  clots  or  semi- 
gelatinous  masses.  It  may  cover  the  bottom  of  the  vessel 
or  occasionally  cause  the  whole  mass  to  gelatinize.  The 
fibrin  can  be  filtered  from  the  liquid  through  muslin  and, 
after  washing,  can  be  tested  (Experiments  77  and  80) .  It  is 
very  similar  to  the  deposit  of  pus  from  fermenting  urine. 
The  pus,  however,  can  be  thinned  with  water.  The  fibrin 
is  insoluble. 

GLYCOSURIA. 

Glucose  is  not  normally  found  in  large  amounts  in 
the  urine,  although  traces  are  frequently — and  perhaps 
always — present.  More  than  a  slight  trace  may  be  re- 
garded as  pathological  if  it  continues  for  any  length  of 
time.  A  transitory  form  of  glycosuria  (alimentary  gly- 
cosuria)  is  often  caused  by  excessive  quantities  of  carbo- 
hydrates, especially  of  sugar  in  the  food.  It  may  be  pro- 
duced by  puncture  of  the  fourth  ventricle  of  the  brain, 
by  injuries  of  the  pancreas,  by  a  number  of  medicinal  sub- 
stances which  act  upon  the  vasomotor  nerves  of  the  liver, 
such  as  phloridzin,  etc. 

The  urine  is  generally,  though  not  always,  of  a  high 
specific  gravity  (1.030  to  1.050),  having  a  light  color  and 
a  whey-like  odor.  The  daily  volume  may  be  increased  to 
ten  times  the  normal,  the  solids  being  likewise  increased. 
When  poured  or  shaken  it  retains  the  foam  for  a  consid- 
erable time. 


GLUCOSE   TESTS.  185 

388.— Test  diabetic  urine  with 

1.  Trommels  test  (26). 

2.  Fehling's  test  (27). 

3.  Bismuth  subnitrate  test  or  Nylandar's  test   (30). 

4.  Phenyl-hydrazin  test  (31). 

5.  Fermentation  test   (32). 

Notice  that  the  other  urinary  constituents  may  mod- 
ify the  results  obtained  with  the  solution  of  pure  glucose. 

For  the  detection  of  glucose  in  urine  the  tests  above  given 
may  be  employed.  No  one  of  these,  however,  is  an  absolute  proof 
of  the  presence  of  glucose.  Other  constituents  of  urine  have  a 
slight  reducing  power,  and  may  respond  to  the  tests  with  alkaline 
solutions  of  copper  or  bismuth,  where  the  action  is  that  of  re- 
duction, for  example,  uric  acid  and  its  salts;  creatinin,  mucin,  and 
others  occurring  in  smaller  amounts  have  this  power  of  reduction 
and  will  reduce  Trommer's  and  Fehling's  reagents.  The  same 
is  true  of  many  medicines  which  pass  into  the  urine.  Trommer's 
and  Fehling's  tests  are  very  sensitive  under  ordinary  conditions, 
but  they  may  fail  in  some  decomposing  urines,  the  ammonia  which 
is  present  keeping  the  cuprous  oxid  in  solution.  Long  boiling  will 
expel  the  ammonia,  and  the  test  may  then  succeed.  Large  amounts 
of  uric  acid,  creatinin,  or  albumin  may  act  in  the  same  manner, 
keeping  the  red  oxid  from  precipitating.  It  must  be  borne  in  mind 
that  the  earthy  phosphates  will  always  be  precipitated  when  the 
urine  is  made  alkaline,  and  consequently,  appear  in  many  of  the 
glucose  tests.  They  are  always  colorless,  as  can  be  shown  by 
washing  them  on  a  filter,  whereas  the  oxid  of  copper  or  bismuth 
reduced  by  the  sugar  are  colored. 

In  the  test  with  the  subnitrate  of  bismuth  the  salt  is  not  so 
easily  reduced  by  other  compounds  than  glucose.  Consequently 
there  is  not  so  much  danger  of  mistaking  these  for  sugar.  With 
a  very  large  excess  of  the  alkali  this  reduction  may  occur.  This 
is  said  not  to  be  the  case  with  Nylander's  modification  of  the  test 
(Experiment  30).  Albumin  is,  however,  decomposed  under  such 
circumstances,  giving  a  black  precipitate.  It  must,  therefore,  be 
removed  from  the  solution  before  the  test  is  made.  With  this  test 


186  THE   URINE. 

very  small  quantities  of  sugar  can  be  detected.  Many  medicinal 
substances  pass  into  the  urine  and  react  with  this  test  also. 

The  phenyl-hydrazin  test  is  not  affected  by  the  reducing 
matters  of  the  urine,  but  it  gives  a  similar  precipitate  with  milk- 
sugar.  Pure  phenyl-hydrazin  must  be  used.  If  it  is  the  hydro- 
chlorid,  the  crystals  should  be  white,  not  brown. 

The  fermentation  test  is  not  very  sensitive.  It  may  be  inter- 
fered with  by  the  presence  of  some  drugs  which  stop  the  action 
of  the  yeast.  If  the  urine  is  not  acid,  it  should  be  made  faintly 
so  with  tartaric  acid.  It  can  be  used  to  distinguish  between  glu- 
cose and  lactose.  Barfoed's  test  (Experiment  28)  may  be  employed 
for  the  same  purpose. 

389. — Determine  the  quantity  of  sugar  in  diabetic 
urine,,  using  Fehling's  solution  (Experiment  33).  Dilute 
the  urine  with  a  measured  volume  of  water  if  necessary, 
and  use  in  the  burette^  as  was  done  in  the  case  of  the  pure 
glucose  solution. 

ACETONE,  (CH3)2CO. 

Normally  acetone  is  present  in  the  urine  only  in 
traces.  Pathologically  it  occurs  there  in  severe  diabetes, 
in  fevers,  in  inanition,  and  cachectic  conditions,  as  well 
as  in  psychoses.  In  diabetes  it  often  is  a  precursor  of  the 
more  dangerous  diacetic  acid.  It  appears  to  be  formed  by 
the  decomposition  of  albuminous  compounds,  and  it  can 
be  produced  in  the  urine  by  the  use  of  a  diet  of  such 
substances.  It  is  a  colorless  liquid  of  a  fruity  odor,  which 
boils  at  56.5°  C.  and  which  can  consequently  be  readily 
distilled  from  the  urine.  The  examination  of  the  urine 
should  be  made  while  it  is  fresh. 

If  a  large  quantity  of  acetone  is  present  in  urine  the 
latter  may  be  tested  directly.  For  small  amounts  it  is  best 
to  distill  from  about  a  liter  one-fourth  of  its  volume  after 
slightly  acidifying  with  sulphuric  acid.  Place  the  distillate 


ACETONURIA.  DIACETURIA.  187 

in  a  retort  and  distill  from  it  about  30  cubic  centimeters. 
This  latter  portion  contains  most  of  the  acetone. 

390.  LIEBEN'S  TEST. — To  a  solution  of  acetone  add 
a  little  sodium  hydrate,  then  a  solution  of  iodin  in  potas- 
sium iodid  and  warm.  lodoform  is  produced  as  a  yellow- 
ish powder  having  a  characteristic  odor.  After  a  time  it 
may  form  six-sided  plates,  which  can  be  seen  with  a  micro- 
scope. Notice  also  the  odor.  Alcohol  gives  the  same  result. 

391. — Prepare  mercuric  oxid  by  precipitating  a  little 
mercuric  chlorid  with  sodium  hydrate.  Wash  by  decanta- 
tion  and  filter  and  wash.  Add  this  to  some  of  the  acetone 
solution,  shake,  and  filter.  The  presence  of  acetone  is 
shown  by  its  dissolving  the  oxid.  This  can  be  proved  by 
pouring  a  layer  of  ammonium  sulphid  solution  on  top  of 
the  filtrate  in  a  narrow  test-tube,  when  the  mercury  will 
be  precipitated  as  a  black  ring  between  the  two  liquids. 

392.  LEGAI/S  TEST. — To  the  liquid  containing  ace- 
tone add  a  drop  of  a  freshly-prepared  solution  of  sodium 
nitro-prussid  and  make  alkaline  with  sodium  hydrate.  A 
ruby-red  color  is  produced.  In  a  few  minutes  it  changes 
to  yellow.  If  it  is  acidified  with  acetic  acid  a  carmin  or 
purplish-red  color  appears  when  much  acetone  is  present. 
On  long  standing  (forty-eight  hours)  this  changes  to  blue. 
(Compare  with  the  results  from  WeyPs  test  for  creatinin 
[Experiment  318],  which  normally  appears  in  the  urine.) 
In  cases  of  doubt  the  urine  may  be  distilled  and  the  ace- 
lone  sought  in  the  distillate.  Creatinin  is  non-volatile. 

DIACETURIA. 

Diacetic  or  aceto-acetic  acid  (CH3COCH2C02H) 
never  appears  normally  in  the  urine,  but  is  found  under 
the  same  pathological  conditions  as  acetone.  In  the  fevers 


188  THE   URINE. 

of  childhood  it  is  not  so  dangerous,  but  with  adults  it 
signals  the  approach  of  coma,  of  which  it  is,  perhaps,  the 
cause,  through  lowering  the  alkalinity  of  the  blood.  Di- 
acetic  acid  is  a  colorless,  strongly-acid  liquid,  soluble  in 
water  and  ether.  On  heating  it  decomposes  below  100° 
to  acetone  and  carbon  dioxid: — 

CH3COCH2C02H  ==  CH3COCH3  +  C02. 

With  ferric  chlorid  it  gives  a  violet-red  solution,  which 
disappears  on  standing  twenty-four  hours  and  more  quickly 
upon  boiling.  This  reaction  can  be  used  to  detect  diacetic 
acid  in  the  urine.  A  number  of  other  substances — like 
salicylic  and  carbolic  acids,  antipyrin,  and  the  acetates — 
give  a  somewhat  similar  reddish  color.  These  are  stable 
at  ordinary  temperatures,  and  only  that  from  the  acetates 
is  decomposed  by  boiling.  The  test  should  be  made  upon 
urine  which  has  been  comparatively  freshly  passed. 

393. — Test  fresh  urine  for  diacetic  acid  by  adding, 
drop  by  drop,  a  solution  of  ferric  chlorid  as  long  as  a 
precipitate  forms.  This  is  ferric  phosphate,  formed  from 
the  phosphates  of  the  urine.  Filter,  and  to  the  filtrate  add 
a  few  drops  more  of  ferric  chlorid.  The  diacetic  acid  gives 
a  violet-red  color.  Allow  it  to  stand  several  hours,  and 
notice  that  it  fades  and  disappears. 

394. — If  this  violet-red  color  was  obtained,  boil  an- 
other portion  of  urine  for  five  to  ten  minutes,  and  after 
cooling  repeat  the  test.  If  the  red  color  was  caused  by 
diacetic  acid  none  will  be  obtained  in  this  second  test,  since 
the  acid  will  have  been  decomposed  by  boiling. 

LACTOSURIA. 

Milk-sugar  may  be  found  in  the  urine  of  women 
toward  the  end  of  pregnancy  and  a  short  time  after  child- 


LACTOSURIA.     CHOLURIA.  189 

birth.  Its  presence  indicates  the  absorption  of  the  sugar 
from  the  fluid  in  the  mammary  gland.  It  may  appear 
with  the  interruption  of  nursing  or  from  stagnation  of 
the  milk  in  the  gland.  When  the  gland  is  well  developed 
and  lactose  is  found  in  the  urine  during  the  period  of 
nursing  it  shows  merely  that  the  secretion  of  milk  is 
abundant.  The  chemical  reactions  of  lactose  are  very 
similar  to  those  of  glucose.  The  principal  differences  are 
that  lactose  ferments  with  yeast  with  difficulty  or  not  at 
all,  and  that  its  power  of  reduction  is  less  than  that  of 
glucose.  Still,  the  distinction  between  the  two  as  they 
occur  in  urine  is  a  matter  of  some  difficulty. 

395. — Try  the  fermentation  test  with  compressed 
yeast,  as  in  Experiment  32,  upon  urine  containing  glucose 
and  that  containing  lactose,  and  notice  that  the  former 
ferments,  with  the  evolution  of  carbon  dioxid,  and  the 
latter  does  not. 

396. — Try  Barfoed's  test  (Experiment  28)  upon  the 
two  kinds  of  urine,  and  notice  that  it  responds  to  glucose, 
but  not  to  lactose.  In  this  test  it  must  be  borne  in  mind 
that  the  other  reducing  substances  of  normal  urine — urates, 
creatinin.,  etc. — may  cause  a  reduction  of  the  copper  salt. 

CHOLURIA. 

In  examining  the  urine  for  bile  two  classes  of  com- 
pounds are  sought  for:  the  biliary  acids  and  the  biliary 
pigments.  The  biliary  acids  do  not  normally  occur  in 
urine,  except  in  small  amounts.  The  pigments  are  more 
commonly  found.  In  the  freshly-passed  urine  usually 
only  bilirubin  is  present,  but  by  oxidation  it  may  be 
changed  to  biliverdin,  etc.  Urine  which  contains  bile  is 
generally  of  a  yellowish-  to  greenish-  brown  color,  and  the 


190  THE    URINE. 

sediment,  if  it  contains  epithelial  cells,  is  often  colored 
brown.  Upon  shaking  the  urine  the  foam  is  yellow  or 
greenish. 

A  common  cause  for  the  appearance  of  the  biliary 
constituents  in  the  urine  is  the  obstruction  of  the  bile- 
duct.  This  may  be  either  from  some  abnormal  growth 
or  merely  from  inflammation  in  the  passages.  The  bile 
is  then  absorbed  by  the  lymphatics  and  excreted  through 
the  kidneys.  The  same  result  may  be  produced  by  any 
abnormal  condition  of  the  liver  which  interferes  with  the 
free  passage  of  the  bile.  A  part  of  the  bile  may  pass  from 
the  blood  into  the  tissues,  manifesting  itself  there  by  its 
characteristic  color  (icterus). 

The  biliary  coloring  matters  may  be  formed  in  the  liver,  but 
they  can  also  be  produced  by  the  decomposition  of  the  haemo- 
globin in  the  blood  and  the  other  tissues  of  the  body,  and  may 
pass  from  here  directly  into  the  urine.  In  this  case  the  urine 
would  contain  none  of  the  biliary  acids,  since  they  do  not  appear 
to  be  formed  outside  the  liver.  A  large  amount  of  these  acids 
with  the  pigments  in  the  urine  indicates  that  the  bile  comes  from 
the  liver  (hepatogenous  icterus).  Some  authors  have  described  as 
a  distinct  form  of  icterus  that  in  which  the  biliary  pigments  ure  de 
rived  from  the  blood-coloring  matters  (haematogenous  icterus).  It 
seems,  however,  to  be  certain  that  the  biliary  acids  may  be  absent 
from  the  urine  even  when  it  contains  bile  from  the  liver  or  gall- 
bladder. 

397.  THE  PRODUCTION  OF  ARTIFICIAL  JAUNDICE. — Insert  a 
small  cannula  into  the  common  bile  duct  of  an  anaesthetized 
albino  rabbit.  Allow  a  dilute  solution  of  indigo  carmine  to  flow 
into  this  from  a  burette.  The  conditions  are  similar  to  those 
where  the  bile  is  reabsorbed  in  consequence  of  some  obstruction  in 
the  common  duct.  In  a  few  minutes  the  mucous  membranes  show 
the  blue  color  and  it  soon  is  seen  under  the  skin  in  all  parts  of  the 
body.  Make  an  autopsy,  examining  the  internal  organs  to  learn 
how  extensive  is  the  diffusion  of  the  color  through  the  tissues. 


BILE    TESTS.  191 

398. — Test  biliary  urine  for  the  pigments  by  slowly 
adding  urine  from  a  pipette,  to  yellow,1  concentrated 
nitric  acid  in  a  test-tube.  The  acid  remains  in  the  bottom, 
and  between  the  liquids  are  seen  the  colored  rings,  as  in 
Experiment  285. 

399. — To  2  to  3  cubic  centimeters  of  Hammarsten's 
reagent  add  a  few  drops  of  urine  and  shake:  a  green  or 
bluish-green  color  results  if  bilirubin  is  present.  With 
minute  amounts  of  bilirubin  or  when  the  urine  is  dark 
colored,  first  precipitate  the  pigments  with  a  little  barium 
chlorid,  allow  it  to  settle,  pour  off  the  liquid,  and  stir  the 
precipitate  with  1  cubic  centimeter  of  the  reagent.  The 
supernatant  liquid  is  green,  converted  by  increasing 
amounts  of  the  acid  mixture  or  by  yellow  nitric  acid 
through  blue  and  violet  to  red  and  yellow. 

400. — If  the  urine  contains  much  bilirubin,  shake  a  large 
test-tubeful  or  more  of  urine  with  half  an  inch  of  chloroform;  pour 
off  the  urine  and  let  the  chloroform  evaporate  on  a  watch-glass. 
The  bilirubin  is  left  in  small,  red  prisms.  It  may  be  purified  by 
dissolving  in  chloroform,  filtering,  and  again  evaporating.  These 
crystals  give  the  play  of  colors  when  moistened  with  nitric  acid. 
They  also  dissolve  in  alkalies,  and  the  solution  becomes  green  on 
standing  (biliverdin). 

401. — If  the  urine  is  dark  colored  from  much  urobilin  or 
blood-coloring  matters  so  that  the  colored  rings  do  not  show,  test 
it  with  Huppert's  test.  Shake  a  test-tubeful  of  the  urine  with  a 
small  amount  of  milk  of  lime,  then  immediately  pass  into  the 
liquid  a  stream  of  carbon  dioxid  to  remove  excess  of  lime.  When 
it  is  neutral,  filter  and  wash  the  precipitate,  which  contains  the 
biliary  pigments.  Moisten  the  precipitate  on  the  paper  with  a 
drop  of  moderately-strong,  yellow  nitric  acid  and  observe  the  play 
of  colors,  from  red  to  green. 


1  The  yellow  acid  can  be  made  by  allowing  the  colorless  acid 
to  stand  for  some  time  in  a  strong  light. 


192  THE    URINE. 

402.— In  urine  which  is  highly  colored  with  other  substances 
the  bilirtibin  may  be  identified  by  Stokvis's  test.  To  20  or  30 
cubic  centimeters  of  urine  in  a  test-tube  add  5  or  10  cubic  centi- 
meters of  a  20-per-cent.  solution  of  zinc  acetate.  Wash  the  pre- 
cipitated bilirubin  upon  a  small  filter;  then  dissolve  it  by  the 
addition  of  a  few  drops  of  ammonia.  The  liquid  which  passes 
through  the  filter  becomes,  after  standing,  brownish  green,  and 
shows  the  spectrum  of  bilicyanin:  an  absorption-band  between  G 
and  D  and  one  between  D  and  E.  If  much  bile  is  present  the 
liquid  becomes  blue  upon  slightly  acidifying. 

403. — To  the  urine  add  a  few  drops  of  very  dilute  tincture  of 
iodin.  A  green  color  results.  If  the  iodin  is  flowed  on  to  the 
top  of  the  urine  by  slanting  the  tube  or  by  dropping  from  a 
pipette  a  green  ring  is  formed. 

404. — Test  the  biliary  urine  for  biliary  acids  by  dis- 
solving in  it  a  few  crystals  of  cane-sugar,  then  dipping  in 
it  a  strip  of  filter-paper.  Dry  the  paper  and  place  on  it 
a  drop  of  concentrated  sulphuric  acid.  In  a  few  seconds 
it  becomes  violet,  best  seen  by  holding  it  before  a  window. 
Too  much  sugar  gives  a  brown  color. 

405. — Instead  of  using  concentrated  acid  make  the 
test  with  dilute  H2S047  as  in  Experiment  268. 

It  is  not  advisable  to  depend  upon  Pettenkofer's  test  alone  in 
the  urine,  as  other  substances  may  be  present  and  give  reactions 
similar  to  those  of  the  bile-acids,  although  their  spectra  are  differ- 
ent. The  pure  bile-acids  may,  in  cases  of  doubt,  be  extracted  by 
the  following  method:  — 

406. — If  the  urine  is  highly  colored  or  only  a  slight  amount 
of  bile-acids  are  present,  it  may  be  necessary  to  extract  the  latter 
before  testing.  Add  to  the  urine  lead  acetate  solution  and  a  few 
drops  of  ammonia  to  make  it  slightly  alkaline.  Wash  with 
water  the  precipitate,  which  contains  the  acids,  then  dry  it.  Ex- 
tract it  several  times  with  warm  alcohol,  filtering  hot.  Make  the 
filtrate  alkaline  with  sodium  carbonate,  and  evaporate  to  dryness 
on  a  water -bath.  Dissolve  the  sodium  salts  of  the  bile-acids  from 
the  residue  with  hot,  strong  alcohol  and  filter.  The  bile-salts  can 


H^IMOGLOBINURIA   AND   H^MATURIA.  193 

be  precipitated  by  adding  ether  to  the  cooled  alcohol.  They 
become  crystalline  on  standing,  or  they  can  be  tested  for  im- 
mediately in  the  alcoholic  filtrate  with  Pettenkofer's  or  other  tests. 


ILEMOGLOBINURIA  AND  H^MATURIA. 

The  haemoglobin  is  found  in  the  urine  in  two  forms: 
first,  dissolved,  no  corpuscles  being  present  (haemoglobi- 
nuria),  and,  second,  in  the  corpuscles  (haematuria). 

The  color  of  urine  which  contains  blood  is  usually 
some  shade  of  red,  but  may  be  dark  brown  or  even  greenish 
brown  when  the  hemoglobin  has  been  changed  to  met- 
hgemoglobin.  Very  small  quantities  may  not  be  detected 
by  the  eye.  The  liquid  is  often  more  or  less  cloudy  from 
corpuscles  and  casts.  There  may  be  enough  blood  present 
to  cause  coagulation  either  in  the  urinary  passages  or  after 
the  urine  is  passed. 

The  free  haemoglobin  is  produced  by  the  destruction 
of  the  corpuscles.  This  may  be  due  to  an  injection  of 
substances  which  dissolve  the  corpuscles,  to  the  trans- 
fusion of  blood,  to  the  action  of  some  poisons  and  in  cer- 
tain infectious  diseases,  like  typhus,  also  after  severe  burns. 
In  this  case  the  urine  should  be  tested  for  haemoglobin. 
If  there  is  a  sediment  the  microscope  reveals  no  corpus- 
cles. 

Haematuria,  where  corpuscles  are  present,  is  more 
common.  It  is  due  to  haemorrhage  in  some  part  of  the 
urinary  tract.  The  corpuscles  appear  as  a  sediment  and 
are  usually  not  in  rolls.  They  may  be  shriveled  or  swollen 
from  standing  in  the  urine.  If  the  hasmorrhage  is  from 
the  kidney,  the  blood  is  usually  well  mixed  with  the  urine 
and  of  a  reddish-brown  color,  the  reaction  being  acid. 
Blood-casts  may  be  present,  and  if  they  are  it  is  a  proof 


194  THE   URINE. 

of  a  renal  haemorrhage.  This  may  occur  in  Bright's  dis- 
ease, also  with  malignant  renal  growths  or  renal  calculi. 

If  the  haemorrhage  is  from  the  bladder  the  urine  is 
often  alkaline,  and  clots  of  blood  are  common.  It  may  be 
caused  by  vesical  calculi,  by  cystitis  or  villous  growths,  and 
by  carcinoma. 

407. — Add  a  very  little  blood  to  highly-colored  nor- 
mal urine,  and  notice  that  the  bands  of  oxyhgemoglobin 
are  visible  through  the  spectroscope,  although  to  the  eye 
there  may  be  no  indication  of  its  presence.  If  the  urine 
is  too  turbid  to  examine  with  the  spectroscope,  it  should 
be  filtered,  and  if  the  residue  is  reddish  on  the  paper  this 
should  be  washed  with  5  cubic  centimeters  of  water  and 
the  washings  examined. 

408. — Convert  the  oxyhasmoglobin  into  haemoglobin 
as  in  Experiment  246,  and  notice  that  the  two  bands 
change  to  one. 

409. — To  urine  containing  a  small  amount  of  blood 
add  enough  sodium  hydrate  to  make  alkaline,  and  heat 
to  boiling.  The  phosphates  of  the  alkaline  earths  will  be 
precipitated,  and  the  precipitate  will  be  colored  reddish  by 
the  haematin  from  the  decomposed  haemoglobin.  If  no 
blood  were  present  the  precipitated  phosphates  would  be 
white.  This  test  will  detect  very  small  amounts  of  blood 
in  urine.  If  the  liquid  is  very  dark  colored,  it  may  be 
necessary  to  filter  and  wash  the  precipitate  before  its  color 
can  be  determined. 

410. — Examine  microscopically  the  sediment  from  a  urine 
after  recent  haemorrhage.  Observe  the  presence  of  red  corpuscles 
and  also  the  change  in  their  form  which  takes  place  after  standing. 

The  guaiacum-hydrogen  peroxid  test  (249)  can  also  be  em- 
ployed, but  its  fallacies  should  not  be  lost  sight  of. 


MUCINURIA.  195 


MUCINUBIA. 

Both  normal  and  pathological  urine  often  contain  a 
substance  which,  although  similar  to  true  mucin,  yet  dif- 
fers from  it  in  many  respects.  On  account  of  this  re- 
semblance it  is  often  called  urinary  mucin.  The  latest  in- 
vestigations indicate  that  it  is  a  nucleoalbumin.  In  nor- 
mal urine  it  appears  after  standing  as  a  light,  fleecy  cloud 
in  the  middle  of  the  liquid.  Its  origin  is  the  mucous  mem- 
brane,— principally  that  of  the  bladder,  ureter,  and  vagina. 
In  small  amounts  it  has  no  special  significance.  In  catar- 
rhal  inflammation  of  the  bladder  it  is  abundant.  In  cys- 
titis and  pyelitis  it  may  give  the  urine  a  gelatinous  appear- 
ance. Mucin  is  also  increased  in  febrile  conditions,  as  well 
as  in  nephritis. 

Urinary  mucin  is  precipitated  from  its  solution  by 
alcohol  or  dilute  acetic  acid  without  heating.  It  may  be 
precipitated  by  very  dilute  mineral  acids,  but  dissolves  in 
excess.  After  precipitation  by  acids  it  is  soluble  in  alka- 
lies. Since  nucleoalbumins,  like  the  mucin  of  urine,  are 
composed  of  an  albuminous  substance  with  a  nuclein,  they 
give  most  of  the  reactions  of  the  albumins,  such  as  those 
with  potassium  ferrocyanid,  picric  acid,  the  biuret  test, 
etc.  Care  is  necessary,  therefore,  to  avoid  confounding 
urinary  mucin  with  small  quantites  of  albumin.  They 
can  be  differentiated  by  the  fact  that  the  mucin  is  precipi- 
tated in  the  cold  by  acetic  acid  even  after  the  urine  has 
been  diluted  with  water,  while  albumin  is  not. 

411. — Dilute  normal  urine  with  its  own  volume  of 
water,  acidify  a  small  beakerful  with  acetic  acid  and  allow 
to  stand  until  the  mucin  has  separated.  Filter  and  wash 
with  water. 


196  THE   URINE. 

412. — Show  that  the  nmcin  dissolves  by  adding  a  few 
drops  of  an  alkali,,  like  sodium  hydrate,  and  that  it  is  re- 
precipitated  from  this  solution  by  acidifying  again  with 
acetic  acid. 

413. — If  urine  containing  much  mucin  can  be  ob- 
tained, apply  the  general  tests  for  protein  and  albumin, 
and  notice  that  it  responds  to  many  of  them. 

LIPURIA,,  OR  CHYLURIA. 

An  abnormal  condition  of  the  urine — not  uncommon  among 
the  inhabitants  of  the  tropics,  but  more  rare  among  those  of  cooler 
climates — is  the  presence  of  fat.  Lipuria,  or  the  appearance  of  fat 
in  the  urine,  may  be  due  to  an  abscess  or  fatty  degeneration  of  the 
kidney;  to  an  excessive  amount  of  fat  in  the  blood,  as  in  preg- 
nancy; or  to  conditions  which  produce  fatty  degeneration  of  other 
organs,  as  the  liver,  and  in  phosphorus  poisoning,  whereby  the 
amount  of  fat  in  the  blood  is  abnormally  increased.  The  chyluria 
of  the  tropics  is  due  to  the  action  of  a  parasite,  which  causes  a 
rupture  of  the  lymph-vessels  and  allows  the  lymph  to  pass  into 
the  urinary  passages.  The  urine  is  often  milky,  and,  on  standing, 
a  creamy  layer  forms.  It  contains  also  the  other  constituents  of 
the  lymph,  albuminous  substances,  etc.  In  cases  of  lipuria  where 
only  a  small  amount  of  fat  is  present  it  may  appear  in  the  form 
of  drops  upon  the  surface,  or  it  may  be  present  in  microscopic 
globules,  either  free  or  in  the  casts  or  epithelial  cells  of  the  sedi- 
ment. The  globules  can  be  perceived  with  the  microscope  and 
separated  by  ether. 

414. — Examine  microscopically  urine  containing  fat. 

415. — To  half  a  test-tubeful  of  urine  containing  fat  add  one- 
fifth  its  volume  of  ether  away  from  the  vicinity  of  a  flame.  Mix 
by  shaking  carefully.  Allow  to  stand  until  the  ethereal  solution 
of  fat  rises  to  the  top.  Notice  that  the  urine  loses  its  milky  ap- 
pearance. Pour  off  the  ether  into  an  evaporating  dish  and  let  it 
evaporate  without  heating.  Dip  a  strip  of  white  paper  in  the 
residue,  and  notice  that  a  greasy  stain  remains  after  drying. 


URINARY   SEDIMENTS.  197 

UKINAKY  SEDIMENTS. 

Besides  the  soluble  constituents  of  the  urine,  there 
are  others  which  appear  as  an  insoluble  deposit  upon  the 
bottom  of  the  containing  vessel  or  floating  in  the  liquid. 
They  may  be  present  in  the  freshly-passed  urine  or  may 
appear  after  a  time.  The  former  are  the  more  important 
to  the  physician,  although  some  conclusions  as  to  the  con- 
dition of  the  system  may  be  drawn  from  the  latter. 

For  the  collection  of  these  sediments  the  best  method 
is  by  the  centrifugal  machine,  or  centrifuge,  this  requir- 
ing so  little  time  that  the  examination  can  be  made  before 
changes  have  occurred  in  any  of  the  constituents.  The 
centrifugal  machine  is  essentially  an  apparatus  where 
tubes  or  other  vessels  can  be  set  in  rapid  rotation.  These 
tubes  swing  from  their  upper  end,  and  as  the  speed  is 
increased  assume  a  horizontal  position.  The  solid  con- 
stituents, being  heavier  than  the  liquid,  are  carried  by  the 
centrifugal  force  to  the  bottom  of  the  tube.  The  tubes 
should  contain  from  15  to  20  cubic  centimeters,  and  be 
rotated  three  to  five  minutes  at  a  speed  of  at  least  1500 
revolutions  per  minute. 

If  a  centrifugal  apparatus  is  not  at  hand,  the  sedi- 
ment is  best  collected  by  allowing  the  urine  to  stand  in 
a  conical  glass  vessel,  containing  4  to  6  fluidounces,  until 
it  has  settled.  Then  decant  the  supernatant  liquid  or 
take,  by  means  of  a  pipette,  a  sample  of  the  sediment  for 
testing. 

Urinary  sediments  can  be  divided  into  two  groups: 
the  organized — or  anatomical — and  the  unorganized — or 
chemical — sediments.  Those  of  the  first  group  are  formed 
by  vital  processes,  and  of  the  latter  by  chemical  force. 


198  URINARY   SEDIMENTS. 

Of  the  unorganized  sediments  some  are  soluble  in  acid 
and  some  in  alkaline  fluids.  Their  presence  depends, 
therefore,  upon  the  reaction  of  the  urine.  They  fall 
naturally  into  two  classes  in  accordance  with  their  solu- 
bility, and  may  be  farther  subdivided  according  to  their 
microscopic  appearance.  The  table  on  the  opposite  page 
gives  the  most  common  varieties. 

Before  examining  the  sediment,  test  with  litmus-paper 
the  reaction  of  the  urine  in  which  it  is  found.  Then  place 
a  drop  of  urine  containing  the  sediment  on  a  glass  slide, 
cover  with  a  cover-glass,  and  examine  microscopically 
with  a  1/2-  or  2/3-rnch  objective.  The  microscopic  ex- 
amination should  be  made  before  the  liquid  evaporates  and 
leaves  on  the  slide  the  soluble  compounds.  A  higher  power 
may  be  used  afterward  if  necessary,  but  generally  the  low 
power  is  preferable.  Chemical  reagents  may  be  applied 
on  the  slide  after  removing  the  excess  of  urine  by  a  piece 
of  porous  paper.  Place  one  drop  of  the  reagent  on  the 
slide  by  the  side  of  the  cover-glass.  It  will  flow  under 
the  cover-glass,  and  its  action  can  be  observed  with  the 
microscope  as  it  comes  in  contact  with  the  different  sedi- 
ments. Care  should  be  taken  not  to  allow  the  reagents 
to  touch  the  microscope-stage.  If  a  low  power  is  used 
without  a  cover-glass  these  tests  may  be  made  in  a  flat 
watch-crystal.  Where  large  quantities  of  a  reagent  are  em- 
ployed, as  in  testing  pus  with  an  alkali,  the  ordinary  chem- 
ical vessels  are  to  be  used. 

Urine  containing  pus  is  turbid  when  freshly  passed, 
and  gives  the  albumin  reactions.  When  much  pus  is  pres- 
ent it  soon  falls  to  the  bottom  as  a  thick  sediment.  Small 
quantities  may  remain  suspended  for  a  long  time.  In  urine 
of  an  acid  reaction  the  pus-corpuscles  can  be  seen.  They 
are  circular  and  colorless,  about  twice  the  diameter  of  the 


CLASSIFICATION. 


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200  URINARY    SEDIMENTS. 

red  blood-corpuscles.  They  appear  granular,  but  when 
brought  in  contact  with  acetic  acid  the  granulation  dis- 
appears and  the  nuclei,  of  which  there  are  two  or  three, 
become  visible.  (Plate  III,  13.)  When  the  urine  becomes 
alkaline,  either  by  fermentation  or  by  the  addition  of  a 
fixed  alkali,  the  corpuscles  disappear  and  the  mass  be- 
comes very  sticky  and  gelatinous,  so  that  it  can  be  drawn 
out  by  a  glass  rod  into  long  threads.  The  turbidity  of 
urine  which  contains  pus  resembles  that  from  urates  or 
from  the  earthy  phosphates.  It  does  not  disappear,  how- 
ever, like  the  former,  by  warming,  nor,  like  the  latter,  upon 
the  addition  of  acids. 

The  source  of  the  pus  in  the  urine  may  be  anywhere 
in  the  urinary  tract.  When  it  is  from  the  kidney  the  urine 
is  apt  to  be  acid  in  reaction,  and  round-celled  epithelium 
or  casts  may  be  present.  When  it  is  from  the  bladder  the 
urine  is  usually  alkaline.  It  may  be  due  to  simple  inflam- 
mation or  to  some  deep-seated  affection  of  the  tissues. 

416. — Examine  microscopically  urine  containing  pus. 
Remove  the  excess  of  liquid  around  the  cover-glass  by 
means  of  a  piece  of  filter-paper.  Put  a  drop  of  acetic  acid 
on  the  slide  and  let  it  run  under  the  cover-glass.  Notice 
the  change  in  the  appearance  of  the  corpuscles. 

417. — Show  that  the  turbidity  does  not  disappear 
upon  warming  or  upon  acidifying. 

418. — Make  Donne's  test  for  pus  by  allowing  it  to 
settle,  then,  after  decanting  off  the  urine,  making  it  alka- 
line with  sodium  hydrate.  The  mass  becomes  extremely 
viscid,  as  is  shown  by  stirring  or  pouring. 

419. — Show  that  the  pus  responds  to  the  albumin  re- 
actions. 

Mucus  as  a  sediment  is  in  the  form  of  a  slimy,  viscid 
liquid,  sometimes  showing  the  mucous  corpuscles.  Its  sig- 


EPITHELIUM.  201 

nificance  lias  been  explained  before.  It  can  be  made  more 
visible  by  adding  a  little  tincture  of  iodin,  which  colors  it 
brownish.  The  addition  of  acetic  acid  to  the  urine  precipi- 
tates nrncin  as  a  fibrous  mass. 

The  epithelial  cells,  being  continually  thrown  off 
from  mucous  surfaces,  are  normally  present  in  small  num- 
bers in  the  urine.  In  such  cases  they  are  usually  from  the 
bladder  and  urethra,  and,  in  women,  from  the  vagina.  A 
large  increase,  however,  is  indicative  of  a  diseased  condi- 
tion of  some  part  of  the  urinary  system.  The  cells  from 
different  parts  of  the  system  are  not  all  of  the  same  shape. 
(Plate  III,  14.)  They  may  be  considered  as  belonging  to 
three  classes:  the  squamous,,  or  pavement-epithelium;  the 
round  celled;  and  the  long,  or  spindle-celled,  epithelium. 
The  squamous  epithelium  is  composed  of  large,  flat,  some- 
what irregular  cells  with  a  distinct  nucleus.  They  may  be 
found  singly  or  united,  like  the  stones  of  a  pavement. 
They  occur  chiefly  in  the  outer  layers  of  the  mucous  mem- 
brane of  the  vagina  and  bladder.  The  round-celled  epi- 
thelium has  smaller  cells  with  a  nucleus  and  nucleolus 
and  are  found  especially  in  the  tubules  of  the  kidneys. 
They  are  also  found  in  the  deeper  layers  of  the  mucous 
membrane  of  other  tissues,  such  as  the  bladder,  urethra, 
and  pelvis  of  the  kidney.  They  are  somewhat  larger  than 
the  pus-corpuscles,  and  the  nucleus  can  be  seen  without 
clearing  by  acetic  acid.  The  long-celled  epithelium  is  nar- 
row and  somewhat  irregular,  with  a  nucleus  visible  with- 
out staining.  They  are  found  in  the  outer  layer  of  the 
membrane  of  the  renal  pelvis  or  in  the  deep  layers  of  the 
bladder,  ureters,  and  urethra. 

Although  the  presence  of  a  single  kind  of  epithelial 
cells  in  the  urine  may  give  an  indication  of  their  origin, 
still  their  occurrence  in  different  tissues  often  renders  this 


202  URINARY   SEDIMENTS. 

a  matter  of  doubt.  The  condition  of  the  cells,  however, 
may  furnish  information  of  the  pathological  changes  which 
have  taken  place.  If  they  appear  disintegrated  or  contain 
fat-globules,  their  origin  is  from  the  locality  of  some  de- 
generative process,  often  of  a  chronic  nature. 

Blood-corpuscles  are  not  normal  in  urine.  In  freshly- 
voided  urine  they  may  retain  their  normal  shape, — that  of 
a  biconcave  disk.  (Plate  III,  13,  d.)  In  acid  urine,  espe- 
cially where  the  specific  gravity  is  high,  they  shrivel  after 
a  time,  the  margins  becoming  irregular.  In  dilute  urine 
and  where  the  reaction  is  alkaline  the  corpuscles  swell, 
and  become  biconvex  or  spherical.  If  there  is  much  blood 
the  liquid  is  reddish,  but  a  slight  amount  may  escape  de- 
tection by  the  unaided  eye.  When  it  is  present  the  albu- 
min reactions  can  always  be  obtained. 

By  urinary  cast  is  meant  an  irregularly-cylindrical 
mass,  composed  of  various  materials,  which  have  been 
formed  in  the  tubules  of  the  kidney,  and  hence  are  of 
about  the  same  size  as  the  tubules.  Opinions  vary  as  to  the 
cause  of  their  formation,  but  most  casts  appear  to  be  due 
to  the  coagulation  of  the  serum  which  passes  into  the 
renal  vessels  owing  to  some  pathological  condition.  The 
presence  of  anatomical  elements — such  as  epithelium,  pus, 
blood,  and  fat — or  their  decomposition  products  in  the 
coagulated  mass  gives  the  different  varieties  of  casts. 

Epithelial  casts  are  not  very  common.  They  consist 
of  cylindrical-shaped  masses  of  round  epithelial  cells  which 
are  thrown  off  from  the  tubules  by  some  pathological  proc- 
ess. The  cells  may  appear  normal  or  they  may  be  more 
or  less  decomposed  and  of  a  granular  appearance,  or  they 
may  contain  minute  fat-globules.  The  cells  sometimes 
seem  to  compose  the  whole  cast  and  sometimes  to  be  scat- 
tered over  its  surface.  (Plate  III,  16.)  When  present, 


CASTS.  203 

they  indicate  inflammation  of  the  kidney.  When  the  cells 
are  degenerated,  the  indications  are  that  the  condition  is 
chronic  or  has  existed  for  some  time. 

Blood-casts  consist  of  coagulated  blood  often  contain- 
ing so  many  red  corpuscles  that  they  are  dark  and  non- 
transparent.  They  may  be  formed  whenever  haemorrhage 
occurs  in  the  urinary  tubules,  and  are  the  best  evidence  of 
this.  They  are  quite  rare,  and  may  be  obscured  under  the 
microscope  by  the  free  blood-corpuscles. 

Casts  of  pus  are  also  very  rare,  but  pus-corpuscles 
are  not  infrequently  seen  in  other  varieties  of  casts. 

By  the  decomposition  and  metamorphosis  of  epithe- 
lium, blood-  or  pus-  cells  in  casts,  the  so-called  granular 
casts  have  their  origin.  They  vary  greatly  in  size,  shape, 
color,  and  in  fineness  of  granulation.  (Plate  III,  15.) 
The  finely  granular  cannot  be  easily  seen  except  with  a 
high  power  of  the  microscope,  although  the  coarsely  granu- 
lar may  be  observed  with  a  low  degree  of  magnification. 
They  often  contain  unaltered  epithelium,  leucocytes,  and 
fat-globules.  Granular  casts  indicate  degeneration  or  a 
long-continued  pathological  condition  of  the  kidney. 

Occasionally  casts  of  fat-globules  are  observed.  They 
result  from  farther  metamorphosis  of  the  granular  casts. 
(Plate  III,  18,  a.) 

In  diseases  of  the  kidney,  like  interstitial  suppurative 
nephritis,  where  bacteria  are  abundant,  casts  composed  of 
these  organisms  are  often  seen.  They  resemble  granular 
casts,  but  are  not  destroyed  by  mineral  acids  and  caustic 
alkalies,  as  are  the  granular  casts.  High  powers  of  the 
microscope  should  be  used  in  their  examination. 

Hyaline  casts  are  almost  transparent  or  at  most  show 
only  a  very  fine  granulation.  On  account  of  their  great 
transparency  they  are  extremely  difficult  to  perceive. 


204  URINARY    SEDIMENTS. 

They  may  be  colored  yellow  by  adding  a  solution  of  iodin. 
In  shape  they  are  usually  long  and  narrow.  Besides  these 
narrow  hyaline  casts,  which  probably  are  formed  in  the 
smaller  tubules,  there  is  sometimes  found  a  broader  vari- 
ety. (Plate  III,  17.)  These  have  an  indented  edge  and, 
in  consequence  of  being  more  highly  refractive,  can  be 
seen  more  easily  than  the  narrow  ones.  They  are  called 
waxy .  casts.  They  often  give  the  amyloid  reaction, — a 
brown  color  with  iodin,  turning  blue  to  violet  upon 
acidifying  with  sulphuric  acid.  They  are  doubtless  formed 
in  the  renal  pyramids.  The  narrow  casts  dissolve  readily 
in  acetic  acid,  but  the  waxy  casts  remain  in  it  for  some 
time.  Hyaline  casts  not  infrequently  have  anatomical  ele- 
ments —  blood-  and  pus-  corpuscles,  epithelium,  etc.  — 
clinging  to  the  surface  or  included  within  the  mass. 

The  origin  of  the  hyaline  casts  seems  to  be  due  to 
the  coagulable  elements  of  the  blood.  It  is  doubtful  if 
they  are  ever  present  in  urine  which  has  not  been  albu- 
minous. Their  presence,  consequently,  is  indicative  of 
the  existence  of  albuminuria.  They  may  be  the  best  evi- 
dence of  such  a  condition  as  interstitial  nephritis,  where 
the  amount  of  albumin  is  small. 

Whatever  variety  of  cast  may  be  present  in  urine,  it 
shows,  without  any  doubt,  that  there  is  a  pathological  con- 
dition of  the  kidney  and  that  the  accompanying  albumin 
is  of  renal  origin. 

Besides  these  cylindrical  casts  there  sometimes  appear 
in  the  urine  the  so-called  cylindroids.  These  are  flat  or 
ribbon-shaped,  rather  than  cylindrical.  They  are  usually 
about  the  diameter  of  casts,  but  longer,  and  resemble  in 
their  transparency  and  solubility  the  hyaline  casts,  their 
composition  being  probably  the  same.  They  are  found  in 
nephritis  and  congestion  of  the  kidneys,  also  in  cystitis. 


BACTERIA.  205 

They  do  not  seem  to  be  characteristic  of  any  pathological 
condition  of  the  kidneys,  but  rather  of  some  irritation  of 
the  lower  urinary  tract  which  has  extended  to  the  kidneys. 

All  casts  are  decomposed  by  bacterial  action.  The 
examination  should,  therefore,  be  made  as  soon  as  possible 
after  the  urine  is  passed  and  the  casts  have  settled.  This 
time  may  be  shortened  to  five  minutes  by  the  use  of  the 
centrifuge.  Without  this  it  will  be  necessary  to  let  the 
urine  stand  several  hours  or  over  night. 

420. — To  examine  urine  for  casts  a  few  drops  from 
the  sediment  obtained  from  standing  in  a  conical  glass 
or  from  the  centrifuge  is  placed  upon  the  microscope- 
slide;  one  with  a  shallow  cell  on  top  is  best.  Cover  it 
with  a  cover-glass  and  remove  liquid  outside  by  filter- 
paper.  Focus  on  the  sediment,  using  a  l/5-ijich  objective, 
then  cut  off  nearly  all  light  from  below.  When  trans- 
parent or  hyaline  casts  are  sought  for  swing  the  mirror  to 
one  side  and  upward  and  throw  the  illumination  upon  the 
slide  obliquely  or  use  a  small  diaphragm.  They  will  be 
more  plainly  visible  by  this  means  than  with  a  strong  illu- 
mination. After  the  casts  have  been  detected  their  cylin- 
drical shape  can  be  shown  by  inclining  the  stage  of  the 
microscope  so  that  they  roll  in  the  liquid. 

BACTERIA. 

The  freshly-voided  normal  urine  contains  no  bacteria. 
They  may  be  present,  however,  under  abnormal  condi- 
tions, and  will  soon  appear  in  normal  urine  .upon  its  stand- 
ing exposed  to  the  air.  On  account  of  the  large  amounts 
of  organic  matter  dissolved  in  the  urine,  it  furnishes  a 
medium  in  which  micro-organisms  readily  grow.  This 
occurs  even  in  the  bladder  if  they  are  introduced  from 


206  URINARY   SEDIMENTS. 

the  outside,  as,  for  example,  by  means  of  an  unclean  cath- 
eter. Urine  containing  bacteria  is  cloudy  and  is  not  cleared 
by  filtration. 

The  non-pathogenic  organisms  are  found  in  putrefy- 
ing or  decomposing  urine.  This  is  usually  not  acid  and 
often  is  strongly  ammoniacal.  They  may  be  found  thus 
in  the  urine  of  cystitis  where  ammoniacal  fermentation  is 
excessive.  Some  of  these  are  of  large  size  and  can  be  ob- 
served with  a  Y5-inch  objective  without  staining.  (Plate 
II,  8.)  The  pathogenic  organisms  are  such  as  the  pus- 
organisms,  the  diplococcus  of  gonorrhoea,  and  also  the 
bacillus  of  tuberculosis  and  the  organisms  of  infectious 
diseases.  They  can  be  examined  and  isolated  by  the  com- 
mon bacteriological  methods. 


SPERMATOZOA. 

These  may  be  found  in  the  urine  of  males  after  coitus 
or  pollution.  They  may  be  present  in  some  diseases,  like 
typhoid,  and  are  constantly  found  in  spermatorrhoea.  By 
straining  during  defecation  there  may  be  a  slight  emis- 
sion of  semen,  and  consequently  the  spermatozoa  be  mixed 
with  the  urine.  They  are  readily  recognized  by  their 
characteristic  shape  under  the  microscope, — a  flattened  oval 
head  united  with  a  long  thread-like  body  and  tail.  (Plate 
III,  18,  c.)  They  are  most  abundant  in  the  first  and  last 
portions  of  the  urine. 

In  freshly  voided  urine  they  may  have  some  motion, 
but  this  soon  ceases.  Acids  and  alkalies,  as  well  as  pure 
water,  stop  it  immediately.  Spermatozoa  resist  putrefac- 
tion and  the  action  of  chemical  reagents,  even  that  of 
strong  acids  or  alkalies. 


URIC  ACID  AND  URATES.  207 

URIC  ACID  AND  URATES. 

The  properties  of  these  compounds  have  been  given 
before.  As  a  sediment,  the  free  acid  and  its  salts  differ 
from  all  others  in  being  colored  yellow  to  brown.  They 
are  not  abnormal  in  urine  unless  they  are  present  as  solids 
when  the  urine  is  passed,  or  are  deposited  within  a  few 
hours,  since  normal  urine  throws  down  uric  acid  on  fer- 
mentation. The  precipitation  of  these  compounds  is 
largely  effected  by  a  concentration  or  an  increase  in  the 
acidity  of  the  urine.  The  normal  or  dibasic  urates  are 
readily  soluble  in  water,  and  do  not  occur  in  sediments. 
When  the  acidity  of  the  liquid  is  increased,  either  by 
fermentation  or  by  the  addition  of  an  acid,  half  the  base 
is  taken  from  these  salts,  leaving  the  monobasic  or  acid 
urates,  which  are  soluble  with  much  more  difficulty.  If  the 
acidity  becomes  still  greater,  all  the  base  is  removed,  leaving 
the  free  acid,  which  is  only  very  slightly  soluble  in  water. 
Of  course,  a  decrease  in  the  volume  of  water  would  be  ac- 
companied by  a  corresponding  increase  in  precipitated 
uric  acid  and  its  compounds.  Hence  a  sediment  of  these 
may  appear  in  the  urine  without  signifying  that  an  in- 
creased quantity  has  been  formed  in  the  body.  Thus,  they 
are  common  in  fevers,  when  the  urine  is  of  small  volume 
and  concentrated.  Less  uric  acid  is  formed  in  the  body 
with  a  vegetable  diet  than  with  one  of  meat. 

Uric  acid  and  urates  as  sediments  occur  mostly  in 
acid  urine  and  can  be  usually  identified  microscopically. 
(Plate  II,  11.)  The  color  is  characteristic.  The  acid  is 
always  crystallized,  commonly  oval  or  diamond  shaped, 
sometimes  visible  to  the  naked  eye,  often  in  clusters  or 
rosettes.  The  urates  are  commonly  salts  of  sodium,  potas- 
sium, or  ammonium.  They  may  be  amorphous  when  ex- 


208  URINARY   SEDIMENTS. 

amined  with  high  powers.  The  so-called  "brick-dust" 
sediment  is  a  mixture  of  the  sodium  and  potassium  urates. 
Sodium  urate  is  also  found  in  fan-shaped  clusters  or  irregu- 
lar groups  of  fine  crystals,  and  sometimes  in  granules. 
(Plate  II,  8.)  Ammonium  urate  makes  up  the  " thorn- 
apple"  crystals:  brown,  spherical  masses  covered  with 
curved  spicules.  (Plate  II,  9.)  The  urates  can  be  differ- 
entiated from  other  sediments  by  being  soluble  on  gently 
warming  the  liquid,  as  well  as  in  alkalies.  The  urates,  as 
well  as  the  free  acid,  give  the  murexid  test  (Experiment 
354).  Uric  acid  is  especially  important  when  found  as  a 
sediment,  from  its  tendency  to  form  calculi.  The  same  is 
true,  to  a  less  extent,  of  the  urates. 

CALCIUM  OXALATE. 

This  salt  is  most  frequent  in  acid  urine.  It  may  exist 
in  two  forms:  the  crystalline,  or  "envelope  shaped/'  and 
the  "dumb-bell  shaped."  Its  appearance  under  the  micro- 
scope affords  the  best  method  of  identification.  (Plate  II, 
10.)  The  crystalline  form  consists  of  octahedral  crystals. 
They  are  never  large,  often  being  smaller  than  a  red  blood- 
corpuscle.  When  sufficiently  magnified,  they  have  some- 
what the  appearance  of  the  back  of  a  square  envelope,  the 
crossed  lines  being  formed  by  the  angles  of  the  crystal. 
In  the  shape  of  the  crystals  they  resemble  some  forms  of 
triple  phosphate,  from  which  they  can  be  distinguished  by 
their  insolubility  in  acetic  acid  and  by  their  smaller  size. 
The  amorphous  form  of  calcium  oxalate  is  disk  shaped, 
with  a  contraction  on  opposite  sides,  so  that  it  somewhat 
resembles  a  dumb-bell.  Calcium  carbonate  has  much  the 
same  form,  but  dissolves  in  acids  with  effervescence. 
Calcium  oxalate  is  insoluble  in  acetic,  but  soluble  in  hydro- 


PHOSPHATES.  209 

chloric  acid.    The  dumb-bell  form  gives  rise  to  calculi  of 
the  bladder. 

Oxalic  acid  and  its  salts  are  found  in  many  fruits  and 
vegetables, — like  tomatoes,  celery,  rhubarb,  etc., — and 
when  these  are  eaten  it  appears  as  the  calcium  salt  in 
the  urine.  It  is  also  produced  in  the  body  from  certain 
foods, — as  from  large  quantities  of  nitrogenous  foods  or 
from  the  carbohydrates,  where  the  oxidation  is  not  com- 
plete. A  small  amount,  then,  may  be  normal,  and  if  it  is 
transitory  is  of  no  great  consequence.  If  the  excretion  is 
continual  it  is  due  probably  to  some  constitutional  weak- 
ness. 

PHOSPHATES. 

The  phosphates  of  the  alkalies,  being  readily  soluble 
in  water,  do  not  appear  as  urinary  sediments.  The  phos- 
phates of  calcium  and  magnesium  are  insoluble  in  water 
or  alkalies,  although  they  dissolve  in  acids.  They,  conse- 
quently, appear  as  sediments  whenever  the  urine  becomes 
alkaline,  but  are  not  found  in  acid  urine  unless  the  acid 
reaction  is  very  faint.  They  can  be  distinguished  from 
other  urinary  sediments  by  dissolving  in  acetic  acid  with- 
out effervescence. 

Triple  phosphate,  NH4MgP04,  is  a  salt  of  phosphoric 
acid  having  two  bases, — ammonium  and  magnesium.  When 
it  is  made  by  precipitating  a  phosphate  by  ammonia  and 
magnesium  sulphate  the  crystals  are  usually  stellate  or 
snow-flake  formed.  As  it  is  made  in  the  urine,  however, 
they  are  more  commonly  in  the  form  of  rhombic  prisms. 
The  terminations  of  the  prisms  are  commonly  truncated; 
so  that  the  crystals  have  a  shape  which  approaches  that 
of  the  end  of  a  coffin,  and  this  gives  rise  to  the  common 
appellation:  "coffin-lid  crystals."  (Plate  II,  8.)  The 

14 


210  URINARY   SEDIMENTS. 

angles  may  not  be  so  truncated  and  the  long  axis  of  the 
crystal  may  be  so  much  shortened  that  it  assumes  the  form 
of  an  octahedron,  like  the  calcium  oxalate.  Unlike  the 
latter,  it  is  soluble  in  acetic  acid.  Calcium  phosphate  in 
the  urine  is  usually  amorphous,  and  always  colorless.  It 
is  formed  when  the  urine  becomes  alkaline  in  the  absence 
of  ammonia.  To  the  unaided  eye  it  resembles  pus,  but 
differs  from  it  in  its  solubility  in  acids.  In  acid  urine  the 
acid  phosphate,  CaHP04,  may  crystallize  in  long  prisms, 
usually  in  clusters.  Tribasic  calcium  phosphate,  Ca3- 
(P04)2,  is  colorless  and  amorphous.  (Plate  II,  7.) 

The  presence  of  phosphates  may  be  due1;o  an  excessive 
formation  in  the  body,  and  they  are  then  usually  accom- 
panied by  systemic  disturbances.  Alkalinity  of  the  urine 
causes  their  appearance  when  there  is  no  excess.  This  may 
be  from  the  food  or  medicine,  from  an  increase  in  the  alka- 
linity of  the  blood,  or  from  fermentation.  Excessive  men- 
tal work  is  often  accompanied  by  phosphatic  sediments. 
Their  long-continued  presence  may  excite  fear  of  the 
formation  of  calculi.  Their  temporary  appearance  is  a 
matter  of  no  grave  significance.  In  urine  which  has  stood 
for  a  time  after  its  passage  they  are  the  most  common  of 
the  sediments. 

421. — Drop  ammonia  into  normal  urine  until  it  is 
slightly  turbid,  and  after  it  has  settled  examine  the  sedi- 
ment with  the  microscope.  It  is  a  mixture  of  the  amor- 
phous calcium  phosphate  and  crystalline  triple  phosphate. 
To  obtain  a  larger  amount  of  the  latter  add  to  the  urine 
a  little  magnesium  sulphate  before  it  is  made  alkaline. 

422. — Precipitate  sodium  phosphate  with  magnesium 
sulphate  after  making  alkaline  by  ammonia.  Notice  the 
difference  in  the  shape  of  these  stellate  crystals  under  the 
microscope  and  those  usually  formed  in  the  urine.  Try 
the  solubility  of  both  forms  in  acetic  acid. 


CALCIUM  SALTS.     TYROSEN.          211 

423. — Make  normal  urine  alkaline  with  sodium  hy- 
drate and  examine  the  precipitated  calcium  and  magne- 
sium phosphates  with  the  microscope.  Try  their  solubility. 

CALCIUM  SULPHATE. 

This  does  not  often  occur  as  a  sediment.  It  may  be 
found  in  acid  urines  as  long  prisms  united  in  clusters. 
(Plate  II,  12,  a.) 

424. — Prepare  crystals  of  calcium  sulphate  by  pre- 
cipitating a  rather  dilute  solution  of  calcium  chlorid  with 
a  few  drops  of  sulphuric  acid.  Dissolve  the  precipitate  in 
boiling  water,  filtering  hot  if  all  does  not  dissolve.  It  will 
reprecipitate  upon  cooling.  Examine  with  the  microscope. 

CALCIUM  CARBONATE. 

This  compound  is  often  found  in  alkaline  urine  with 
calcium  phosphate.  It  appears  as  a  sandy  powder  which, 
when  examined  microscopically,  is  seen  to  consist  of 
spherical  bodies  formed  of  concentric  layers  or  to  have 
the  dumb-bell  shape  of  calcium  oxalate.  (Plate  II,  9.) 
It  dissolves  readily  in  acetic  or  other  acids,  with  the  evolu- 
tion of  carbon  dioxid  gas. 

TYROSIN. 

Tyrosin  is  not  often  found  as  a  sediment  because  of 
its  solubility  in  water,  but  it  sometimes  appears  as  such, 
though  never  in  a  normal  condition  of  the  system.  It 
crystallizes  in  minute  needle-shaped  crystals,  which  are 
usually  aggregated  into  clusters  or  sheaves.  (Plate  II, 
12,  c.)  Its  microscopic  appearance  is  the  best  means  of 
identifying  it.  The  chemical  tests  have  been  given. 


212  SYSTEMATIC   TESTING   OF   URINE. 

Tyrosin  in  the  urine  has  the  same  source  as  in  diges- 
tion— the  decomposition  of  protein  compounds.  It  is  im- 
probable that  it  comes  from  the  intestine,  but  from  other 
parts  of  the  system.  It  is  indicative  of  retrograde  met- 
amorphosis of  the  nitrogenous  tissues.  Thus,  it  is  present 
in  acute  atrophy  of  the  liver,  in  suppurative  processes,  and 
in  phosphorus  poisoning,  which  is  accompanied  by  degen- 
eration of  the  liver.  Leucin  is  often  found  at  the  same 
time.  (Plate  II,  12,  ft.) 

FAT. 

The  appearance  and  significance  of  fat  in  the  urine 
(lipuria)  has  already  been  discussed. 

* 

SYSTEMATIC  TESTING  OF  TJKINE. 

In  the  systematic  testing  of  urine  the  course  is  often 
varied,  as  the  symptoms  may  point  to  the  likelihood  of 
the  presence  or  absence  of  certain  substances.  The  quan- 
titative tests  may  be  made  use  of  or  not  according  to 
circumstances.  The  following  are  the  determinations 
which  are  most  important,  with  the  tests  which  may  be 
employed : — 

1.  Amount  passed  in  twenty-four  hours. 

2.  Color  ^  XT         . 

f  Normal  or  abnormal. 

QCy  (If  the  latter,  what  is  the  cause? 

4.  Odor  J 

5.  Chemical  reaction. 

If  alkaline,  is  it  from  NH3  or  fixed  alkalies? 
(Experiment  338). 

6.  Specific  gravity  at  60°  F.  (15.5°  C.). 

7.  Urea:    percentage   and    amount   in    twenty-four 

hours   (Experiment  348  or  349). 


SYSTEMATIC   TESTING   OF   URINE.  213 

8.  Glucose. 

General  test,  Trommels  or  Fehling's  (Experi- 
ments 26  and  27). 

Confirmatory  tests  (Experiments  29,  30,  and  31). 
Quantitative  test,  Fehling's  (Experiment  33). 

9.  Acetone. 

Experiments  390,  391,  and  392. 

10.  Diacetic  acid. 
Experiments  393  and  394. 

11.  Albumin. 

General  test,  heat  and  HN08  (Experiment  380). 
Confirmatory  tests   (Experiments  381,  382,  and 

383). 
Quantitative   test,    Esbach's    (Experiment   384), 

or  weighing. 

12.  Blood. 

General  tests,  spectroscope  (Experiments  245, 
246,  and  247) ;  also  corpuscles  in  sediment. 
Confirmatory  test,  guaiacum  test  (Experi- 
ment 249;  also  Experiment  409). 

13.  Bile-pigments. 

General  test,  colors  with  yellow  HN03  (Experi- 
ment 398). 

Confirmatory  tests  (Experiments  401,  402,  and 
403). 

14.  Bile-acids. 

Experiments  404,  405,  and  406. 

15.  Peptone. 
Experiment  387. 

16.  Organic  sulphates. 
Experiment  374. 

17.  Indican. 
Experiments  376  and  377. 


214  SYSTEMATIC    TESTING    OF    URINE. 

18.  Uric  acid:  amount. 
Experiment  358. 

19.  Total  nitrogen :  percentage  and  amount  in  twenty- 

four  hours. 
Experiment  350. 

20.  Chlorin:   amount. 
Experiments  365  or  366. 

21.  Phosphoric  acid:   amount. 
Experiment  370. 

22.  Identification    of    sediments    if    present.      (Page 

199.) 

I.  UNORGANIZED. 

(A)  Crystalline. 
Uric  acid. 
Calcium  oxalate. 
Calcium  phosphate. 
Triple  phosphate. 
Other  rarer  compounds. 

(B)  Amorphous. 
Urates. 
Phosphates,,  etc. 

II.  ORGANIZED. 

Pus. 

Mucus. 

Blood-corpuscles. 

Bacteria. 

Spermatozoa. 

Epithelium:   kind  and  probable  source. 

Casts:   kind  and  probable  source. 
The  proof  of  the  presence  of  any  abnormal  constit- 
uent should  not  be  allowed  to  rest  upon  one  test,  but  sev- 
eral should  be  tried. 


VARIATIONS    WITH    FOOD.  CALCULI.  215 

425.  THE  EFFECT  OF  FOOD  ON  THE  COMPOSITION  OF  THE 
URINE. — Let  a  number  of  subjects  each  select  food  of  a  different 
class  and  eat  only  this  for  twenty-four  hours,  collecting  all  the 
urine  for  the  period.  The  following  dietaries  will  give  a  variety: 

1.  Largely  animal. 

2.  Vegetable. 

3.  Rich  in  purins,  sweetbreads,  etc. 

4.  Purin  free — milk,  eggs,  wheat  bread,  butter,  cheese. 

5.  Low  in  nitrogen. 

6.  No  food. 

Determine  volume,  specific  gravity,  color,  reaction;  amounts 
of  nitrogen,  iiric  acid,  phosphoric  acid,  urea.  Report  results. 


URINARY  CALCULI. 

The  constituents  of  calculi  are  the  same  as  those  of 
the  chemical  sediments,  and  the  causes  which  give  rise  to 
the  formation  of  the  latter  will  also  favor  the  production 
of  calculi  in  the  bladder.  To  these  various  names  are 
applied,  according  to  their  size:  sand,  gravel,  stone,  and 
calculi,  or  concretions.  They  vary  from  the  microscopic 
to  aggregations  as  large  as  an  orange.  They  are  generally 
not  composed  of  a  single  material,  but  have  at  the  centre  a 
nucleus,  and  this  is  surrounded  by  layers,  often  of  two  or 
more  compounds  in  alternation.  The  nucleus  may  be  a 
mass  of  foreign  matter,  or  it  may  be  a  clot  of  blood  or  a 
particle  of  one  of  the  sediments  around  which  material, 
perhaps  of  a  different  kind,  can  be  deposited.  Uric  acid 
concretions  are  the  most  common.  They  are  brown  in 
color,  rough  of  surface,  and  brittle.  The  form  of  the 
crystals  cannot  be  seen,  but  they  give  the  murexid  test. 
They  dissolve  in  sodium  or  potassium  hydrate,  from  which 
solutions  the  uric  acid  may  be  precipitated  in  the  crystal- 
line form  by  the  addition  of  a  mineral  acid.  Uric  acid 
calculi  are  formed  only  in  an  acid  urine. 


216  URINARY    CALCULI. 

The  urates  are  often  found  mixed  with  the  uric  acid 
deposits  or  with  those  of  calcium  oxalate.  The  ammonium 
salt  is  the  most  abundant.  They  are  generally  small, 
grayish,  and  rather  soft.  They  give  the  murexid  test. 
They  are  deposited  from  acid  urine,  except  the  am- 
monium urate,  which  is  formed  in  an  alkaline  solution. 

Calcium  oxalate  concretions  are  commonly  of  large 
size  and  are  very  hard.  The  surface  is  rough  and  warty. 
They  are  called  "mulberry  calculi"  from  the  resemblance 
of  the  surface  to  that  of  the  fruit.  The  urine  is  generally 
acid,  unless  where  the  presence  of  the  stone  has  produced 
cystitis.  They  are  often  dark  in  color  from  the  blood  which 
has  been  incorporated  with  them. 

The  phosphates  can  only  be  present  in  calculi  when 
the  urine  is  alkaline.  They  are  generally  rather  soft  and 
easily  broken.  Calcium  phosphate  has  a  chalky  appear- 
ance. Triple  phosphate,  NH4MgP04,  is  found  with  other 
substances.  It  is  more  commonly  on  the  outside  of  the 
stone,  being  precipitated  by  the  alkaline  reaction  produced 
by  the  presence  of  the  concretion  in  the  bladder.  A  mix- 
ture of  the  triple  phosphate  and  calcium  phosphate  is  fusi- 
ble with  the  blow-pipe  and  is  known  as  the  "fusible  cal- 
culus/' 

Calcium  carbonate  is  not  common,  although  found 
occasionally. 

The  analysis  of  calculi  is  made  by  the  use  of  chemical 
methods.  The  stone  should  be  broken  or,  better,  if  it  is 
large  enough,  sawed  through  the  middle.  This  shows 
the  layers  of  which  it  is  composed  and  the  nucleus.  If 
there  appears  to  be  any  difference  in  the  layers,  they  should 
be  tested  separately.  Heat  a  piece  upon  platinum  foil  and 
notice  whether  it  fuses  and  whether  it  is  combustible  or 
not.  If  it  fuses  it  is  an  indication  of  a  phosphate  of  cal- 


ANALYSIS.  217 

cium  and  triple  phosphate.    If  it  is  combustible  it  consists 
of  organic  compounds. 

Blackening  when  ignited  is  evidence  of  organic  mat- 
ter, but  if  slight  it  may  be  merely  mucus  arising  from 
irritation  of  the  bladder,  and  not  an  essential  part  of  the 
calculus.  Ignition  on  the  foil  will  divide  the  constituents 
into  two  classes,  although  both  may  be  present. 

COMBUSTIBLE,  OK  INCOMBUSTIBLE,  OR 

ORGANIC.  INORGANIC. 

1.  Uric  acid.  1.  Calcium  phosphate. 

2.  Ammonium  urate.  2.  Calcium  oxalate. 

3.  Calcium  carbonate. 

4.  Triple  phosphate. 

5.  Urates  of  K,  Na,  and  Ca. 

If  it  is  composed  largely  or  entirely  of  organic  matter 
try  the  murexid  test  (Experiment  354)  for  uric  acid  and 
urates.  If  inorganic  compounds  are  present,  powder  a  piece 
and  treat  in  a  test-tube  with  2  or  3  cubic  centimeters  of 
dilute  hydrochloric  acid.  Carbonates  dissolve  with  effer- 
vescence of  carbon  dioxid  gas,  the  others  without.  Warm, 
if  necessary.  Filter,  if  it  does  not  give  a  clear  solution. 
To  one-fourth  of  the  filtrate  in  a  test-tube  add  sodium 
hydrate  until  it  is  alkaline,  and  test  for  ammonia  by  hang- 
ing in  the  tube  a  strip  of  moist  red  litmus-paper,  being 
careful  that  it  does  not  touch  the  side  of  the  tube  which 
is  wet  with  the  sodium  hydrate.  The  tube  can  be  allowed 
to  stand  corked  over  night  or  the  ammonia-gas  can  be  ex- 
pelled from  the  liquid  by  boiling.  If  present  it  will  turn 
the  paper  blue. 

To  the  remainder  of  the  solution  in  hydrochloric  acid 
add  ammonia  until  it  is  alkaline,  acidify  with  acetic  acid, 
and  boil.  If  there  is  a  precipitate,  filter. 


218 


ANALYSIS    OF    CALCULI. 


Precipitate  is  cal- 
cium oxalate.  Test 
after  washing  and 
drying  by  heating 
to  a  bright-red  heat 
on  platinum  foil. 
After  cooling  it 
should  turn  moist 
red  litmus  -  paper 
blue. 


To  the  filtrate  add  ammonium  oxalate, 
boil,  and,  if  there  is  a  precipitate,  filter  Avhile 
hot. 


A  white  precipi- 
tate shows  cal- 
cium, probably 
originally  present 
as  phosphate  or 
carbonate. 


The  filtrate  is  to  be 
tested  for  magnesium 
and  plwsphoric  acid.  For 
Mg  make  one-half  alka- 
line with  ammonia  and 
if  the  liquid  remains 
clear,  add  sodium  phos- 
phate. A  fine,  white 
crystalline  precipitate 
with  either  reagent  in- 
dicates Mg.  For  phos- 
phoric acid  make  re- 
mainder acid  with  strong 
HNO3  and  add  ammo- 
nium molybdate.  A  yel- 
low precipitate  appears. 


Urates  of  K,  Na,  and  Ca  can  be  found  by  boiling  the 
powdered  calculus  in  water,  filtering,  and  testing  the  fil- 
trate by  the  murexid  test.  Or  if  it  is  evaporated  to  dryness 
and  the  residue  is  ignited  on  platinum  the  sodium  and 
potassium  will  remain  as  carbonates,  giving  an  alkaline  re- 
action to  litmus-paper. 


THE  METEIC  SYSTEM. 

In  all  work  in  modern  chemistry  the  metric  system 
of  weights  and  measures  is  employed.  The  unit  of  length 
is  the  meter  (39.37  inches);  of  weight  is  the  gramme  (or 
gram),  which  is  the  weight  of  1  cubic  centimeter  of  water 
at  4°;  and,  of  capacity,  the  liter,  which  has  the  volume  of 
1  cubic  decimeter. 


METRIC    SYSTEM.  219 


MEASURES  OP  LENGTH. 

10  millimeters  =  1  centimeter. 

10  centimeters  =  1  decimeter. 

10  decimeters  =  1  meter. 

10  meters  =  1  decameter. 

10  decameters  =  1  hectometer. 

10  hectometers  =  1  kilometer. 


MEASURES  OP  WEIGHT. 

10  milligrammes  =  1  centigramme. 
10  centigrammes  =  1  decigramme. 
10  decigrammes    =  1  gramme. 
10  grammes          =  1  decagramme. 
10  decagrammes  =  1  hectogramme. 
10  hectogrammes  =  1  kilogramme. 


MEASURES  OF  VOLUME. 

10  milliliters  =  1  centiliter. 

10  centiliters  =  1  deciliter. 

10  deciliters  =  1  liter. 

10  liters  =  1  decaliter. 

10  decaliters  =  1  hectoliter. 

10  hectoliters  =  1  kiloliter. 

The  following  are  especially  to  be  remembered: — 

One  gramme  is  the  weight  of  1  cubic  centimeter  of 
water  measured  at  4°  C. 

A  liter  contains  1000  cubic  centimeters  and  a  liter  of 
water  weighs,  therefore,  1000  grammes. 


220  WEIGHTS   AND   MEASURES. 

The  following  are  convenient  in  the  conversion  of  the 
weights  and  measures  of  one  system  into  another: — 

1  meter     =39.37  inches. 

1  foot        =0.304  meter. 

1  liter        =  61.03  cubic  inches  =  1.06  U.  S.  qts. 

1  liter        =33.81  U.  S.  fluidounces. 

1  gramme  =  15.43  grains. 

1  grain      =  0.0648  gramme. 

1  ounce  (apoth.)  =31.1  grammes. 

1  ounce  (avoirdupois)  =28.35  grammes. 

1  pound  (apoth.)          =373.2  grammes. 

1  pound  (avoirdupois)  =  453.6  grammes. 


EEAGENTS. 

FEHLING'S  SOLUTION. — Make  up  and  preserve  in  two 
parts:  A  and  B. 

(A)  Dissolve    34.64   grammes    of   crystallized,    non- 
effloresced  copper  sulphate  (CuS04,  5H20)  in  water  and 
make  up  the  volume  to  500  cubic  centimeters. 

(B)  Dissolve  173  grammes  of  pure,  crystallized  Eo- 
chelle  salt  (sodium  and  potassium  tartrate)  and  50  grammes 
of  sodium  hydrate  in  water,  and  bring  the  volume  to  500 
cubic  centimeters. 

Before  using  mix  equal  volumes  of  A  and  B. 

NYLANDER'S  EE AGENT. — Dissolve  in  100  cubic  centi- 
meters of  water  2  grammes  of  subnitrate  of  bismuth, 
4  grammes  of  Eochelle  salt,  and  10  grammes  of  NaOH. 

ESBACH'S  REAGENT  contains,  in  a  liter,  10  grammes 
of  picric  acid  and  20  grammes  of  citric  acid. 


REAGENTS.  221 

OBERMAYER'S  REAGENT  FOR  INDICAN  contains  2  to  4 
grammes  of  ferric  chlorid  in  a  liter  of  concentrated  hydro- 
chloric acid. 

MILLON'S  EEAGENT. — Dissolve  1  part  of  mercury  in 
2  parts  of  nitric  acid  (sp.  gr.,  1.42),  first  at  ordinary  tem- 
perature, then  with  the  aid  of  heat.  When  it  has  dis- 
solved add  twice  its  volume  of  water,  and  after  several 
hours  decant  the  reagent  from  any  sediment  that  may  be 
present. 

HAMMERSTEN'S  REAGENT  FOR  BILE  PIGMENTS  consists 
of  1  volume  of  nitric  acid  and  19  volumes  of  hydrochloric 
acid,  each  25  per  cent.  When  it  has  become  yellow  by 
standing,  dilute  with  four  times  its  volume  of  alcohol. 

BARFOED'S  REAGENT  contains  acetic  acid  and  cupric 
acetate  of  such  a  strength  that  there  shall  be  about  1  per 
cent,  of  each  when  mixed  with  the  sugar  solution  to  be 
tested. 

GUNZBURG'S  EEAGENT  FOR  HC1. — Dissolve  2  grammes 
of  phloroglucin  and  1  gramme  of  vanillin  in  100  cubic 
centimeters  of  alcohol. 

BOAS'S  EEAGENT  FOR  HC1. — Dissolve  5  grammes  of 
resorcin  and  3  grammes  of  cane-sugar  in  100  cubic  centi- 
meters of  dilute  alcohol. 

Methyl-violet     ^ 

A  solution  in  water  containing  about  1 
Trorjseolin  00 

per  cent,  of  the  coloring  matter. 
Alizarin  sodium 

sulphonate      t 

Phenolphthalein,  a  1-per-cent.  alcoholic  solution. 
Dimethyl-amido-azobenzene,  a  0.5  per  cent,  alcoholic 
solution. 


222 


REAGENTS. 


lodin,  a  1-per-cent.  solution  of  potassium  iodid  in 
water  with  a  few  crystals  of  iodin. 

Of  the  common  reagents,  the  following  strengths  may 
be  conveniently  used: — 

Barium  chlorid  ^j 

Ammonium  hydrate 

A  .         y,     . ,     >  10  per  cent,  in  water. 

Ammonium  chlorid     { 

Tannic  acid  J 

Ammonium  oxalate 
Ammonium  molybdate 
Potassium  ferrocyanid 
Potassium  ferricyanid 
Lead  acetate 
Sodium  phosphate 
Ferric  chlorid 
Cupric  sulphate 
Mercuric  chlorid 
Sodium  hydrate 
Silver  nitrate 


5  per  cent,  in  water. 


Picric  acid 


A  saturated  solution  in 


Lime-water,  or  calcium  hydrate  J       water. 

Sulphuric  acid,  10  per  cent.;  pour  1  volume  into  18 
volumes  of  water. 

Nitric  acid,  10  per  cent.;  1  volume  of  acid  and  6  of 
water. 

Hydrochloric  acid,  5  per  cent.;  1  volume  of  acid  and 
6  of  water. 

Acetic  acid,  6  per  cent. 


INDEX. 


Absorption  from  stomach,  test, 

91. 

Acetic  acid  in  stomach,  73. 
Aceto-acetic  acid,  187. 
Acetone  in  urine,  186. 
Acid  albumin,  46. 

from  digestion,  78 
Acid  phosphates  in  gastric  juice, 

73,  82. 
tests  for,  83,  87. 

in  urine,  170. 

Acidity  of  gastric  juice,  deter- 
mination, 85. 

urine,  determination,  152. 
Acrolein,  28. 
Albuminates,  46. 
Albuminoids,  58. 
Albuminous  substances,  34. 

classification,  38. 

in  urine,  178. 
Albumins,  39. 

in  urine,  178. 

limitations      of      tests, 

94 

Albuminuria,  178. 
Albumose    from    digestion,    78, 
99. 

in  urine,  182. 

Alcohol,  production,  13,  16. 
Alkali  albumin,  47. 
Alkaline  phosphates,  169. 
Alloxuric  bases,  136. 
Ammonium  urate,  163. 
Amphoteric  reaction,  142. 
Amylopsin,  99. 


Amyloses,  2. 

Animal  body,  composition,  1. 

charcoal,  132. 
|    Anti  compounds,  49. 

Babcock's  test  for  fat  in  milk, 

145. 
Bacteria  as  ferments,  62. 

in  urine,  205. 
Bacterial  casts,  203. 
|   Barfoed's  test,  15. 
Bile,  122. 

-acids,  123. 

in  urine,  189,  192. 
preparation,  127. 
salts,  preparation,  125. 
tests  for,   130,   191. 
-pigments,  124. 
in  urine,  191. 
preparation,  129. 
tests  for,  130,  191 
Biliary  calculi,  128. 

mucin,  122. 
Bilifuscin,  125. 
Biliprasin,  125. 
Bilirubin,  124. 
Biliverdin,  124. 
Biuret  test,  37. 
Blood,  101. 
-casts,  203. 

coagulation,  102,  105. 
-corpuscles,  101. 

determination  of  num- 
ber, 102. 
isolation,  104. 

(223) 


224 


INDEX. 


Blood-fibrin,  106. 

in  urine,  193,  202. 

-plasma,  101. 

-reaction,  101. 

-serum,  101,  106. 

specific  gravity  of,  101. 
determination,  104. 

-stains,  tests  for,  121. 
Boettger's  test,  15. 
Bone,  132. 
Bone-black,  132. 
Brain,  141. 
Butter-fats,  143. 
Butyric  acid,  90. 

in  stomach,  73. 

preparation,  65. 

tests,  90. 

Calcium  carbonate  in  urine,  211. 

oxalate  in  urine,  208. 

sulphate  in  urine,  211. 
Calculi  of  bladder,  215. 

analysis,  216. 
Cane-sugar,  20. 
Carbohydrates,  1. 

classification,  2. 

reactions,  22. 
Carbonic  oxid  haemoglobin,  114, 

117,  118. 
Carnin,  135. 
Cartilage,  132. 
Casein,  55,  143. 

preparation,  54. 
Casts  in  urine,  202. 

bacterial,  203. 

blood,  203. 

epithelial,  202. 

fatty,  203. 

granular,  203. 


Casts  in  urine,  hyaline,  203. 

pus,  203. 

waxy,  204. 
Cellulose,  11. 
Cerebrin,  142. 
Cheese,  55,  143. 
Chlorin  in  urine,  167. 
Cholalic  acid,  123,  127. 
Cholesterin,  123. 

preparation,  128,  142. 
Choluria,  189. 
Chyluria,  196. 
Chyme,  75. 

Coagulated  albumin,  51. 
Coagulation,  36,  43. 
Coffin-lid  crystals,  171. 
Collagen,  58,  131. 
Colloid,  4. 
Connective  tissues,  131. 

digestion  of,  75. 
Creatin,  135,  141. 
Creatinin,  135,  141. 

separation  from  urine,  167. 
Crystalloid,  4. 
Cylindroids  in  urine,  204. 
Cystin,  47. 

Dextrin,  8. 
Dextrose,  12. 
Diacetic  acid,  187. 
Diaceturia,  187. 
Dialysis,  4. 
Digestion,  gastric,  75. 

pancreatic,  94,  96. 

salivary,  67. 
Disaccharids,  2. 

Earthy  phosphates,  169. 

in  urinary  sediments,  209. 


INDEX. 


225 


Egg-albumin,  crystallization,  42. 

purification,  41. 
Elastin,  61. 
Emulsion,  23. 

Enterogenic  peptonuria,  182. 
Enzymes,  62. 
Epithelial  casts  in  urine,  202. 

cells  in  urine,  201. 
Essential  oils,  25. 
Extractives,  135,  139. 
Fat  in  urine,  196. 

in  milk,  determination,  143, 

145. 
Fats,  23. 

digestion  of,  97. 
Fatty  acids,  24. 

casts,  203. 

Fehling's  test,  14,  17. 
Fermentation,  62. 

in  stomach,  72. 

in  urine,  148,  151. 

of  glucose,  16. 
Fibrin,  51,  89. 

in  urine,  184. 

preparation,  102. 
Fibrinuria,  184. 
Foods,  composition  of,  1. 

Gall-stones,  125. 
Gastric  digestion,  75,  77. 
juice,  71. 

collection,  81. 
preparation     of    artifi- 
cial, 77. 
testing,  81. 
tests,  interpretation,  92. 

limitations  of,  92. 
Gelatin,  59. 
Globulins,  44. 


Globulins  in  urine,  182. 
Glucose,  12. 

in  urine,  184. 

preparation  of  pure,  14. 

tests,  14,  15,  16. 
in  urine,  185. 

limitations,  185. 
Glycerin,  formation,  23. 
Glycocholic  acid,  123. 
Glycocoll,  123,  166. 
Glycogen,  8  . 
Glycosuria,  184. 
Glycuronic  acid,  22. 
Gmelin's  test,  130. 
Granular  casts,  203. 
Granulose,  4. 
Grape-sugar,  12. 
Guaiacum  test  for  haemoglobin. 

117. 
Guanin,  135,  136. 

Hsematin,  107,  114. 
Hsematogen,  56,  58. 
Hsematogenous  icterus,  190. 
Haematoporphyrin,  107,  115. 

preparation,  120. 
Hsematuria,  193,  202. 
Haemin,  107,  114,  118. 
Haemochromogen,  107,  115. 

preparation,  121. 
Haemoglobin,  107. 

derivatives,  107. 

quantitative  determination, 
109. 

spectrum,  102,  108,  116. 
Haemoglobinuria,  193. 
Haines's  test,  14. 
Heart-burn,  73. 
Hemi  compounds,  49. 


226 


INDEX. 


Hepatogenous  icterus,  190. 
Hippuric  acid,  165. 
Huppert's  test,  130. 
Hyalin  casts,  203. 
Hydrochloric    acid    of    gastric 

juice,  72. 
tests,  84,  85. 
Hypoxanthin,  135,  136. 

Icterus,  190. 

Indican,  174,  176. 

Indigo,  174. 

Indol,  174. 

Indoxyl,  174. 

Inorganic  sulphates,  173. 

Inversion,  3. 

Invertin,  63. 

Invert-sugar,  20. 

Iron  of  animal  body,  source,  57. 

Isotonic  solution,  102. 

Kephyr,  19. 

preparation,  66. 
Keratin,  61. 
KjehldahPs     determination     of 

nitrogen,  159. 
Koumiss,  19. 

Lactalbumin,  144. 
Lactic  acid,  fermentation,  prep- 
aration, 65. 
in  stomach,  72. 
tests,  88. 
sarco-lactic,  137. 
Lactose,  19. 

in  urine,  188. 
preparation,  19. 
quantitative  test,  145. 
tests,  20.  189. 


Lacosturia,  188. 

Laking,  101. 

Lanolin,  124. 

Lead  plaster,  24,  28. 

Lecithin,  30. 

Leucin,  from  digestion.  95,  97. 

in  urine,  212. 

preparation,  99. 

tests,  100. 
Lipuria,  196. 
Lithic  acid,  161. 
Liver-starch,  8. 
Liver-sugar,  8. 

Maltose,  21. 
Malt-sugar,  21. 
Methsemoglobin,  107,  113. 

preparation,  120. 
Metric  system  of  weights  and 

measures,  218. 
Milk,  142. 
Milk-sugar,  19. 

in  urine,  188. 
Millon's  test,  39. 
Monosaccharids,  2. 
Mucin,  52. 

in  urine,  195,  200. 
Mucinuria,  195. 
Mucoids,  52. 
Murexid  test,  164. 
Muscular  -tissues,  134. 
Muscle  plasma,  135. 

preparation,  138. 

-serum,  139. 
Myelin,  30. 
Myogen,  138. 
Myosin,  45,  138. 

Nitrogen,  quantitative  determi- 
nation, 159. 


INDEX. 


227 


Nuclein  bases,  56,  136. 
Nucleins,  56. 
Nucleoalbumins,  53. 
Nylander's  test,  15. 

Oils,  25. 

Olein,  25. 

Olive-oil,  30. 

Organic  sulphates,  173. 

preparation,  175. 
Organized  ferments,  62. 
Ossein,  59. 
Oxyhaemoglobin,  107,  111. 

preparation,  119. 

spectrum,  112,  116. 

Palmitic  acid,  preparation,  28. 

Palmitin,  25. 

Pancreatic,  digestion,  94,  96. 

juice,  93. 
Paranucleins,  56. 
Pentoses,  21. 
Peptone,  48. 

in  urine,  181. 
Pepsin,  73,  77. 

preparation,  64,  75. 

test,  90. 

valuation,  80. 
Pettenkofei-'s  test,  126. 
Phenyl-hydrazin  test,  15. 
Phosphates  in  calculi,  216. 

gastric  juice,  73,  83. 

urine,  169,  209. 

Phosphoric  acid,  determination, 
171. 

source,  170. 
Phosphorized  fats,  30. 
Plasma,  blood-,  101. 

muscle-,  135. 
Polysaccharids,  2. 


Protagon,  141. 
Proteins,  32. 

classification,  34. 

compound,  51. 

crystallization,  42. 
Proteoses,  48. 
Ptyalin,  69. 

preparation,  69. 
Purdy's  test,  18. 
Purin,  136. 

bases,  136. 
Pus-casts,  203. 

in  urine,  198. 
Pyogenic  peptonuria,  183. 

Reagents,  220. 
Rennin,  74. 

preparation,  78. 

test,  79. 
Rigor  mortis,  135,  137. 

Saliva,  66. 

pathological,  68. 
Saponification,  23. 

in  digestion,  97. 
Sarcin,  135. 

Sediments  in  urine,  197. 
Sjoqvist's  quantitative  test  for 

HC1,  86. 
Soaps,  24,  27. 

Sodium    hypobromite,    prepara- 
tion, 158. 

urate,  163,  208. 
Spermatozoa,  206. 
Starch,  4. 

tests,  5,  6. 
Steapsin,  97. 
Stearin,  25. 
Sucrose,  20. 
Sulphates  in  urine,  172. 


228 


INDEX. 


Sulphocyanates,  in  saliva,  67. 
Syntonin,  45. 

Tanning,  59. 
Taurin,  123. 

preparation,  128. 
Taurocholic  acid,  123. 
preparation,  127. 
Test-meal,  81. 
Tests,  Arnold's  for  lactic  acid, 

89. 
Babcock's  for  fats  in  milk, 

145. 

Barfoed's  for  sugar,  15,  19. 
Biuret   for   albumins,    etc., 

37. 

Boettger's  for  sugar,  15. 
Esbach's  for  albumin,  179, 

181. 

Fehling*s  for  sugar,  14,  17. 
Gmelin's  for  bilirubin,  130. 
Guaiacum  for  blood,  117. 
Haines's  for  glucose,  14. 
Heller's  for  albumin,  181. 
Huppert's  for  bilirubin,  130. 
Jaffa's  for  indican,  177. 
KjehldahTs    for    nitrogen, 

159. 

Legal's  for  acetone,  187. 
Lieben's  for  acetone,  187. 
Millon's  for  albumins,  39. 
Murexid  for  uric  acid,  164. 
Nylander's  for  sugar,  15. 
Obermayer's     for     indican, 

177. 
Pettenkofer's  for  bile  acids, 

126. 
Phenyl-hydrazin  for  sugar, 

15. 
Purdy*s  for  sugar,  18. 


Tests,  Sjoqvist's  for  acidity  of 
gastric  juice,  86. 

Toepfer's  for  acidity  of  gas- 
tric juice,  85. 

Trommer's  for  sugar,  14. 

Xanthroproteic  for  albumi- 
nous substances,  37. 
Toepfer's  test,  85. 
Triple  phosphate,  171. 

in  sediments,  209. 
Trommers'  test,  14. 
Trypsin,  94,  95. 
Tryptophan,  94,  96. 
Tyrosin,  from  digestion,  95,  97. 

in  urine,  211. 

preparation,  99. 

tests,  100. 

Unorganized  ferments,  62. 

urinary  sediments,  199. 
Urates,  163. 

in  calculi,  216. 
in  sediments,  207. 
Urea,  152. 

preparation,     from     urine, 

156. 

synthetic,  157. 
quantitative  determination, 

153,  158. 

Uric  acid,  136,  161. 
in  calculi,  215. 
in  sediments,  207. 
quantitative  determination, 

164. 
Urinary  calculi,  215. 

analysis,  216. 
casts,  202. 
mucin,  195,  200. 
sediments,  197. 


INDEX. 


229 


Urine,  amount,  147. 
color,  148. 
constituents,  146. 
daily  variations,  147. 
fermentation,  151. 
odor,  148. 
reaction,  150. 
specific  gravity,  149. 
systematic  testing,  212. 


Vegetable  parchment,  12. 

Waxy  casts,  204. 

White  fibrous  tissue,  131. 

Xanthin,  135,  136. 
Xanthin  bases,  136. 
Xanthoproteic  reaction,  37. 

Zymase,  63. 
Zymogens,  62,  76. 


DAY    AND    1 
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